Compositions and methods to treat cardiac diseases

ABSTRACT

Phosphonate and phosphinate N-methanocarba derivatives of AMP including their prodrug analogs are described. MRS2339, a 2-chloro-AMP derivative containing a (N)-methanocarba (bicyclo[3.1.0]hexane) ring system in place of ribose, activates P2X receptors, ligand-gated ion channels. Phosphonate analogues of MRS2339 were synthesized using Michaelis-Arbuzov and Wittig reactions, based on the expectation of increased half-life in vivo due to the stability of the C—P bond. When administered to calsequestrin-overexpressing mice (a genetic model of heart failure) via a mini-osmotic pump (Alzet), some analogues significantly increased intact heart contractile function in vivo, as assessed by echocardiography-derived fractional shortening (FS) as compared to vehicle-infused mice. The range of carbocyclic nucleotide analogues for treatment of heart failure has been expanded.

REFERENCE TO CROSS-RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application61/306,687 filed on Feb. 22, 2010, which is incorporated herein byreference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH & DEVELOPMENT

The U.S. Government has certain rights in this invention pursuant toNational Institutes of Health Grant No. RO1-HL48225.

BACKGROUND

The actions of extracellular nucleotides in cell signaling are mediatedby two classes of cell surface purinergic receptors: P2X receptors areligand-gated ion channels activated by extracellular ATP, and P2Yreceptors are G protein-coupled receptors activated by both adenine anduracil nucleotides. In the heart, for example, a variety of P2 receptorsare expressed.

Cardiac P2X receptors represent a novel and potentially importanttherapeutic target for the treatment of heart failure. A P2X receptor onthe cardiomyocyte mediates cardioprotection and is activated by ATP orits potent analogue 2-MeSATP 1, as demonstrated using the calsequestrin(CSQ) model of cardiomyopathy. Extracellular ATP can cause an ioniccurrent in murine, rat, and guinea pig cardiac ventricular myocytes. TheP2X₄ receptor is an important subunit of the native cardiac P2Xreceptor, which mediates ionic current induced by extracellular ATP.This P2X current was up-regulated in cardiac ventricular myocytes of theCSQ hearts. Furthermore, cardiac myocyte-specific overexpression of theP2X₄ receptor can mimic the beneficial effects following chronicinfusion of P2X agonist analogues. This analysis suggested thatregulation of this cardiac P2X receptor is protective in cardiachypertrophy or failure.

(1′S,2′R,3′S,4′R,5′S)-4-(6-amino-2-chloro-9H-purin-9-yl)-1-[phosphoryloxymethyl]bicyclo[3.1.0]hexane-2,3-diol,(MRS2339, 3) is an (N)-methanocarba monophosphate derivative of2-chloro-AMP 2 that contains a rigid bicyclic ring system(bicyclo[3.1.0]hexane) in place of ribose. This ring system impedeshydrolysis of the 5′-phosphate in a model compound by its nucleotidase.Compound 3 induced a current in the CSQ myocyte similar to that bycompound 1, characteristic of the action of the P2X₄ receptor.Chronically administered MRS2339 (compound 3) rescued the hypertrophicand heart failure phenotype in the CSQ-overexpressing mouse. Whenadministered via an Alzet mini-osmotic pump, it significantly increasedlongevity as compared to vehicle-injected mice. The improvement insurvival was associated with decreases in heart weight/body weight ratioand in cross-section area of the cardiac myocytes. Compound 3 was devoidof any vasodilator action in aorta ring preparations indicating that itssalutary effect in heart failure was not due to any vascular unloading.

Activation of this myocyte P2X receptor leads to the opening of anonselective cation channel permeable to Na⁺, K⁺, and Ca²⁺. The currentis inward at negative membrane potentials, reverses near 0 mV, andbecomes outward at positive potentials. The continuous activation ofthis receptor channel under the resting or negative membrane potentialswould produce an inward current while its activation during depolarizedportions of the action potential should lead to an outward current.These ionic currents represent a possible ionic mechanism by which thecardiomyocyte P2X channel achieves its protective effect.

What is needed are additional myocyte P2X receptor activators that havecardioprotection activity.

SUMMARY

In one aspect, disclosed herein are phosphonate and phosphinateN-methanocarba derivatives of AMP comprising

wherein

Q¹ is O or S;

R¹ is hydrogen, optionally substituted alkyl, optionally substitutedcycloalkyl, halogen, or N(R⁶)₂, wherein each R⁶ is independentlyhydrogen, optionally substituted alkyl, or optionally substitutedcycloalkyl;

R² is hydrogen, optionally substituted alkyl, optionally substitutedcycloalkyl, optionally substituted alkynyl, N(R⁶)₂, or halogen;

R³ is hydrogen, optionally substituted alkyl, N(R⁶)₂, or halogen;

R⁴ is hydroxyl, optionally substituted alkyl, optionally substitutedalkoxy, optionally substituted aryl, optionally substituted —Oaryl, orN(R⁶)₂;

R⁵ is hydroxyl, optionally substituted alkyl, optionally substitutedalkoxy, optionally substituted aryl, or optionally substituted —Oaryl;or

-   -   alternatively, R⁴ and R⁵ form a 5- or 6-membered cyclic        structure with the phosphorus atom where the cyclic structure        contains at least two oxygen atoms and at least 2 or 3 carbon        atoms, wherein the carbon atoms are optionally substituted with        alkyl or aryl where the chain is attached; and

Y is a linking group linked to the phosphorus atom by a carbon atom;

or

wherein X is O or S; n is 1, 2, or 3; and R⁷ is optionally substitutedalkyl or optionally substituted aryl;

or

wherein Z is a bond or —O—C(═O)— where the carbonyl carbon is bonded tothe oxygen of the bicycle group and the oxygen is bonded to thephosphorus atom;

or

wherein R⁸ is hydrogen or optionally substituted alkyl; and R⁸ isoptionally substituted alkyl, optionally substituted alkoxy, oroptionally substituted aryl;or

wherein G is O or S—S; and R¹⁰ is hydrogen, hydroxyl, optionallysubstituted alkyl, optionally substituted alkoxy, or optionallysubstituted aryl; or

wherein R¹¹ is hydrogen, optionally substituted alkyl, or optionallysubstituted aryl; or

wherein

Q¹ is O or S;

Q² is O or S;

R¹ is hydrogen, optionally substituted alkyl, optionally substitutedcycloalkyl, halogen, or N(R⁶)₂, wherein each R⁶ is independentlyhydrogen, optionally substituted alkyl, or optionally substitutedcycloalkyl;

R² is hydrogen, optionally substituted alkyl, optionally substitutedcycloalkyl, optionally substituted alkynyl; N(R⁶)₂, or halogen;

R³ is hydrogen, optionally substituted alkyl, N(R⁶)₂, or halogen;

R⁴ is hydroxyl, optionally substituted alkyl, optionally substitutedalkoxy, optionally substituted aryl, optionally substituted —Oaryl, orN(R⁶)₂;

R⁵ is hydroxyl, optionally substituted alkyl, optionally substitutedalkoxy, optionally substituted aryl, or optionally substituted —Oaryl;or

-   -   alternatively, R⁴ and R⁵ form a 5- or 6-membered cyclic        structure with the phosphorus atom where the cyclic structure        contains at least two oxygen atoms and at least 2 or 3 carbon        atoms, wherein the carbon atoms are optionally substituted with        alkyl or aryl where the chain is attached; and        Y¹ is a linking group,

with the proviso that when Q¹ and Q² are both O, and Formula (VII) isnot enriched with deuterium, then R⁴ and R⁵ are not both hydroxyl,

a deuterium enriched isomer thereof, or a pharmaceutically acceptablesalt thereof.

In one aspect, a method of treating a mammalian subject in need oftreatment for a cardiac or vascular disease or condition responsive toactivation of the cardiac and/or vascular P2X receptor comprisesadministering to the subject in need thereof an effective amount of aphosphonate or phosphinate N-methanocarba derivative of AMP for thetreatment for the cardiac or vascular disease or condition responsive toactivation of the cardiac and/or vascular P2X receptor.

In another aspect, a method of improving cardiac contractile performanceand/or cardiac function in a mammal in need thereof comprisesadministering to the mammal in need thereof an effective amount ofphosphonate or phosphinate N-methanocarba derivative of AMP for theimprovement of cardiac contractile performance and/or cardiac function.

In yet another aspect, a method of treating a mammalian subject in needof treatment for a cardiac hypertrophy, systolic heart failure,diastolic heart failure, ischemic cardiomyopathy, non-ischemiccardiomyopathy, or adverse remodeling and injury followingischemia/reperfusion injury, comprises administering to the mammal inneed thereof an effective amount of phosphonate or phosphinateN-methanocarba derivative of AMP.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the beneficial effects of 2-Cl substituted phosphonatederivatives of (N)-methanocarba AMP in heart failure mice. Variousderivatives of phosphonates were dissolved in sterile normal saline (NS)at 3.3 μM and were infused subcutaneously individually via an Alzetminipump in CSQ mice as described in Methods. After 28 days of infusion,the in vivo heart function was assessed using echocardiography-derivedfractional shortening (FS). (A) The 2-Cl substituted 5′-phosphonatederivative 4 was able to improve in vivo cardiac contractile performancein heart failure mice as compared to normal saline-treated heart failureanimals (one-way ANOVA with posttest comparison, P<0.05), while theunsubstituted 5 was ineffective (P>0.05). (B) Similarly, 2-Clsubstituted higher homologue 9 was able to enhance cardiac contractilefunction (P<0.05), while the parent unsubstituted compound 10 did notimprove the contractile function (P>0.05). Data were mean±SE.

FIG. 2 shows that chronic infusion of compound 4 resulted in improvedechocardiographically derived FS in CSQ heart failure mice. Followingchronic subcutaneous infusion of NS or compound 4, two-dimensionaldirected M-mode echocardiography was carried out as described inMethods. The heart rate (HR) is indicated on each figure. RepresentativeM-mode echocardiography was shown for a CSQ animal infused with normalsaline (NS) (A) and for a CSQ mouse infused with compound 4 (B). A heartfrom the NS-infused mice showed less shortening of both septum and LVfree wall than did compound 4-infused mice.

FIG. 3 shows changes in intracellular calcium in 1321N1 humanastrocytoma cells stably expressing the hP2Y₁ receptor. Fluorescence inresponse to a known hP2Y₁ receptor agonist 2-MeSADP (EC₅₀ 10.3±0.4 nMcompound 3 (EC₅₀722±55 nM), or the phosphonate analogues (compounds4-11, all inactive at 10 μM) was quantified using a FLIPR-Tetra.

FIG. 4 shows chronic infusion of compound 11a caused an increased LVcontractile fractional shortening in calsequestrin (CSQ)-overexpressingmodel of heart failure. Following 14 days of infusion of compound 11a(n=6 mice) or normal saline (NS, n=4) in CSQ mice, LV fractionalshortening (FS) was compared between the two groups. Treatment withcompound 11a resulted in improved in vivo LV contractile function inanimals with heart failure. Data are mean and standard error.

FIG. 5 shows chronic infusion of compound 11a resulted in preservationof LV wall thickness in CSQ-overexpressing heart failure mice. The invivo cardiac wall thickening was assessed by echocardiography following14 days of infusion of compound 11a or NS in CSQ mice. a: In compound11a-infused animals, the LV posterior wall thickness during systole(LVPW@S) was greater (P<0.05) than that in NS-infused CSQ mice (P<0.01).b: Similar data were obtained when septal thickness during systole(IVS@S) was compared between compound 11a-treated and NS-treated CSQmice, P<MRS 0.01. c: LV posterior wall thickness during diastole wasalso greater in compound 11a-infused than in NS-infused CSQ mice,P<0.05. Data are expressed as mean±SEM. The data suggest that treatmentwith compound 11a was able to prevent LV wall thinning in heart failure.

The above-described and other features will be appreciated andunderstood by those skilled in the art from the following detaileddescription and appended claims.

DETAILED DESCRIPTION

Cardiac and vascular diseases and conditions responsive to activation ofthe cardiac P2X receptors include cardiomyopathy and those diseasesassociated with defects in cardiac contractility. As agonists of P2Xreceptors, the phosphonate or phosphinate N-methanocarba derivatives ofAMP are particularly useful in the treatment of, for example, cardiachypertrophy, cardiac failure resulting from any cause of abnormal Ca²⁺homeostasis or from myocardial injuries, vascular insufficiency leadingto myocardial infarction, for post-myocardial infarction conditions, forpost-myocardial infarction conditions within the short-termpost-infarction period, and for diastolic heart failure. Agonists of P2Xreceptors can be used as cardioprotective agents to increase survivalrates in individuals who have had a cardiac event such as a myocardialinfarction or to prevent cardiac events in high risk patients. Thephosphonate or phosphinate N-methanocarba derivatives of AMP are alsouseful in the treatment of systolic heart failure of any etiology,ischemic cardiomyopathy, or non-ischemic cardiomyopathy.

Hypertrophy and heart failure, for example, remain a medical conditionwith “unmet medical need”. The available medications have been shown tobe beneficial with only modest reduction in mortality rate. The agentsdisclosed herein are a new class of oral agents that may prolonglifespan in heart failure. They may be more effective than the currentlyavailable medications given beneficial result obtained in the animalmodel.

In one embodiment, the phosphonate or phosphinate N-methanocarbaderivative of AMP is a prodrug analog. As used herein, the term prodrugmeans a compound that is administered in inactive or less active formthat is metabolized in vivo to a more active form. Established methodscan be used to demonstrate that the phosphonate or phosphinatederivatives and/or their analogs are prodrugs. For example, the prodrugcan be injected into mice and the appearance of the parent drug followedby HPLC of serum samples taken at different time points. Because the P2Xreceptors are ion channels on the cell surface that are stimulated byextracellular ligands, a prodrug approach was selected that should allowfor cleavage of the administered compound prior to internalization intoa cell, but not in the stomach or gut.

In another example, the regeneration of the parent drug from the prodrugcan be followed by spiking blood with a solution containing a suspectedprodrug at a concentration of about 20 μM. During incubation the bloodmixture can be maintained at 37° C. and samples removed at 5 min, 0.5hr, 1 hr, 2 hr and 4 hr. After removal the samples are immediatelyhemolyzed in tubes prefilled with ice chilled water and stored at −20°C. until analysis. For analysis, an internal standard consisting of 100μl of a 40 μM DMSO solution of the N6-(fluorenylmethyl) derivative ofadenosine and 100 μl of a 10% solution of sulfosalicylic acid are addedto each sample. After gently vortexing for 5 min, the samples areextracted three times, successively, with 0.5 ml of water-saturatedethyl acetate. Each extraction consists of the addition of ethylacetate, vortexing for 5 min, centrifugation for 5 min at 2000 g, andmanual separation of supernatant with an automatic pipette. The extractfractions are combined and evaporated to dryness under a stream ofnitrogen gas. The residue is then reconstituted in 50 μl of an HPLCmobile phase system. 40 μl of this solution is injected for eachchromatographic run to provide a kinetic profile for conversion of theprodrug into its parent nucleoside. The chromatography is performed, forexample, at room temperature using a reversed-phase column (ZorbaxEclipse 5 μm XDB-C18 analytical column, 250×4.6 mm) equipped with aguard column packed with C-18 material. The flow rate is 1.0 ml/min, andthe detection wavelength of 280 nm is used. The time course for relativeconcentration of each derivative is calculated based on the fractionalpercentage of total nucleoside detected and was plotted.

The inventors have explored the structure activity relationship (SAR) ofphosphonate analogues of compound 3 in a model of cardioprotection.

Although an (N)-methanocarba nucleoside 5′-monophosphate was shown to bea poor substrate of 5′-nucleotidase (CD73), replacement of thephosphoester group of compound 3 with a phosphonate could furtherincrease the in vivo half-life because of the stability of the C—P bond.Phosphonate analogues of nucleotides and other known ligands, in somecases, have been shown to display activity at P2 receptors. In oneembodiment, substitution of monophosphate esters with phosphorothioategroups of various ligands has been found to provide resistance tophosphatase-catalyzed hydrolysis without reducing binding affinity. Inanother embodiment, the introduction of deuterium in place of hydrogenat strategic locations on labile receptor ligands and other drugs hasbeen shown to increase biological lifetime due to an isotope effectwithout reducing binding affinity.

5′-phosphonate and 5′-methyl phosphonates of (N)-methanocarba adenine 4,11 or 2-Cl adenine derivatives 5, 12 were synthesized using thepreviously reported compound 13. At least two synthetic pathways leadingto these target molecules can be envisioned viz. routes A and B (Scheme7A) based on the installation of phosphonate group. Route A involvesphosphonate installation at the nucleoside level to generate keyintermediate 42, which could be used as common intermediate for thegeneration of both phosphonates and methyl phosphonates 43 usingMichaelis-Arbuzov reaction conditions. While route B involvesphosphonate installation at the sugar level on halogenated intermediates17 and 37 to provide intermediates 18 and 38. Route B does not have acommon intermediate, like route A, and, as a result, it involves alonger and more laborious synthetic sequence. Generally, theMichaelis-Arbuzov reaction conditions to generate phosphonatederivatives require long reaction times (24 to 48 hours) at elevatedtemperatures (120 to 180° C.). Moreover, removal of trialkyl phosphitereagent needs high temperatures and high vacuum. Because of these harshconditions, a Michaelis-Arbuzov reaction at the nucleoside levelgenerally results in very poor yields (less than 25% yield) of thedesired phosphonates along with the formation of a dark-colored thermaldegraded products of the nucleosides. On the other hand,Michaelis-Arbuzov reaction at the sugar level generally results in verygood yields. Hence, although route B is time consuming and contains moresynthetic steps than route A, we have decided to obtain thesephosphonate derivatives via route B, believing that it would be reliablewith good yields.

Long chain saturated and unsaturated phosphonates of (N)-methanocarbaadenine or 2-Cl adenine derivatives 7-10 could be achieved from the samestarting compound 13 as for the phosphonates 4, 5, 11 and 12. Similar tothe synthesis of phosphonates 4, 5, 11 and 12, these phosphonates couldpossibly be obtained via two synthetic routes viz, route C and D (Scheme7B), based on the installation of the phosphonate group. The long chainunsaturated phosphonate could be installed by oxidation and 5′-alcoholof either nucleoside 24 or compound 15 followed by the Wittig-typereaction using tetraisopropyl methylenediphosphonate and NaH to providephosphonate diester 26 or 30, respectively. One could expect thisreaction to proceed smoothly at both nucleoside and sugar stages. Sincethe route A has a common intermediate 26 for the synthesis of long chainsaturated and unsaturated 5′-phosphonates 7-10, we have decided toexplore the synthesis of these phosphonates by the shorter syntheticroute C.

There is increasing evidence that chronic activation of the nativecardiac P2X receptor by nucleotide analogues protects against theprogression of heart failure. The P2X₄ receptor is an essential subunitof this native receptor, but we do not know what other P2X subtypes arepresent. The cardiac myoctye receptor is not identical to the vascularP2X₄ receptor, which has a key role in the response of endothelial cellsto changes in blood flow.

The synthetic nucleotide analogue 3 activates the native cardiac P2Xreceptor, as indicated in electrophysiological experiments with normalcardiac myocytes and those that overexpress CSQ and based on its in vivoability to improve the heart failure phenotype of these animals. Therigid carbocyclic ring system contained in this derivative stabilizesnucleotides toward the action of nucleotidases. Therefore, compound 3 isexpected to be more stable than the corresponding riboside. In thepresent study, we have synthesized fully hydrolysis-resistant adenosinemonophosphate derivatives based on phosphonate linkages. The C—O—P bondof compound 3 was found to be stable over 24 hours in aqueous medium atpH 1.5 to simulate the acidity of the stomach, however incubation at 37°C. in the presence of mammalian cell membranes (1321N1 astrocytomacells) resulted in considerable hydrolysis of the 5′-phosphate of 3(data shown). Therefore, a more stable structural alternate to the5′-phosphate linkage was sought.

An in vivo screen of cardiac function was used to test the novelanalogues. Thus, the results of this chronic study likely reflect bothpharmacodynamic and pharmacokinetic factors. Several of the novelphosphonate analogues displayed the same agonist activity as compound 3at native cardiac P2X receptors, i.e. they protected the heart musclewhen chronically administered in the CSQ model. The SAR analysis showedthat considerable cadioprotection was associated with specificstructural features of the phosphonate derivatives. The variation in thechain length and saturation at the 5′ carbon provided consistent resultsin the in vivo screen. Two of the phosphonates, 4 and 9, both saturatedhomologues containing a 2-Cl substitution, improved FS, while theunsaturated phosphonates and 2-H analogues were inactive. The mostfavorable FS (20.25%, compared to 13.78% in controls) was observed for(1′S,2′R,3′S,4′R,5′S)-4′-(6-amino-2-chloropurin-9-yl)-2′,3′-(dihydroxy)-1′-(phosphonomethylene)-bicyclo[3.1.0]hexane4, which is the equivalent of compound 3 in which the 5′-O has beenexcised. The higher homologue 9 displayed a FS of 19.26%. Thus, it ispossible to extend the SAR around compound 3, for chronic activation ofthe cardiac P2X receptor leading to a beneficial effect in heartfailure.

Further, the 5′-phosphate 3 and the prodrug 9a (MRS2944) are stable toacidic conditions representative of stomach acid (pH 1.5). However,incubation of 5′-phosphate 3 or the diester phosphonate 9a at 37° C. inthe presence of mammalian cell membranes (1321N1 astrocytoma cells)resulted in considerable hydrolysis (data shown). This indicates thatthe desirable cleavage of a diester phosphonate prodrug such as 9a isfeasible in the presence of tissue, while the phosphate drugs such as 3might be subject to premature cleavage in vivo. Without being held totheory, it is believed that the prodrug approach will allow the maskeddrug to pass through the stomach to be absorbed intact in theintestines, and for the charged phosphoryl group to remain intact untilthe free drug reaches the site of action in the heart. Therefore, thestable phosphonates would be suitable in this scheme. The cleavage ofthe prodrug derivatives is to occur in circulation prior to reaching thetissue site of action, because intracellular internalization would beundesirable. Therefore, many of the prodrug schemes that aim forpenetration of the masked drug into the cells, i.e. for antiviral oranticancer application of nucleotide derivatives, would likely not besuitable here.

It is not feasible to study the analogues at a recombinant homotrimericP2X₄ receptor system, because the endogenous cardiac P2X receptor isthought to be composed of P2X₄ receptor subunits in heteromericassociation with a yet unidentified P2X subtype. The P2X₄ receptor isknown to associate with other P2X receptor subtypes, and theseheterotrimers are pharmacologically distinct from P2X₄ homotrimers.

Another site of action of adenine nucleotides in cardiac tissue is themetabotropic P2Y₁ receptor, which causes a nitric oxide-dependentrelaxation of the vascular smooth muscle. Therefore, we tested thenucleotides as P2Y₁ receptor agonists to account for the possibilitythat the observed cadiovascular effects of the phosphonate derivativeswere a result of activation of an endothelial P2Y₁ receptor. Compound 3was initially characterized in assays of PLC as a weak hP2Y₁ receptoragonist (EC₅₀ 1.89 μM), and that conclusion is consistent with thepotency observed here in inducing calcium transients in the same cellline. All of the phosphonate derivatives tested were inactive at theP2Y₁ receptor. This suggests the use of these compounds as moreselective pharmacological probes of the endogenous cardiac P2X receptorthan compound 3. However, it is worth noting that the cardioprotectionprovided by compound 3 was shown to be independent of the P2Y₁ receptorby its inability to dilate aortic rings and by use of a P2Y₁-selectiveantagonist MRS2500. This antagonist could not block the membrane currentevoked by 3 under voltage clamp in mouse cardiac myocytes.

Disclosed herein are novel phosphonate or phosphinate N-methanocarbaderivatives of AMP. Suitable N-methanocarba derivatives of AMP are givenin Formula (I) below:

wherein

Q¹ is O or S;

R¹ is hydrogen, optionally substituted alkyl, optionally substitutedcycloalkyl, halogen, or N(R⁶)₂, wherein each R⁶ is independentlyhydrogen, optionally substituted alkyl, or optionally substitutedcycloalkyl;

R² is hydrogen, optionally substituted alkyl, optionally substitutedcycloalkyl, optionally substituted alkynyl, N(R⁶)₂, or halogen;

R³ is hydrogen, optionally substituted alkyl, N(R⁶)₂, or halogen;

R⁴ is hydroxyl, optionally substituted alkyl, optionally substitutedalkoxy, optionally substituted aryl, optionally substituted —Oaryl, orN(R⁶)₂;

R⁵ is hydroxyl, optionally substituted alkyl, optionally substitutedalkoxy, optionally substituted aryl, or optionally substituted —Oaryl;or

-   -   alternatively, R⁴ and R⁵ form a 5- or 6-membered cyclic        structure with the phosphorus atom where the cyclic structure        contains at least two oxygen atoms and at least 2 or 3 carbon        atoms, wherein the carbon atoms are optionally substituted with        alkyl or aryl where the chain is attached; and

Y is a linking group linked to the phosphorus atom by a carbon atom, adeuterium enriched isomer thereof, or a pharmaceutically acceptable saltthereof.

The Y linking group is optionally substituted C₁-C₆ alkylene, optionallysubstituted C₁-C₆ alkenylene, optionally substituted C₁-C₆ alkynylene,or optionally substituted —C₁-C₆ alkylene-O—C₁-C₆ alkylene-. In oneembodiment, Y is —CH₂—. In another embodiment, Y is —CH₂CH₂—. In yetanother embodiment, Y is —CH═CH—.

In one embodiment, R⁴ is hydroxyl, methyl, or C₁-C₆ alkoxy, and R⁵ ishydroxyl, methyl, or C₁-C₆ alkoxy.

In one embodiment, R⁴ and R⁵ are linked together by a 3 carbon chainsubstituted with an aryl group at the 1 position (e.g.,1-aryl-1,3-propanyl cyclic ester group).

In one embodiment, R¹ is NH₂.

In another embodiment, R³ is hydrogen.

In yet another embodiment, R² is hydrogen chloro, iodo, or C₁-C₂alkynyl.

In one embodiment where the N-methanocarba derivative of AMP isaccording to Formula (I), Y is —CH₂— or —CH₂CH₂—, R¹ is NH₂, R² ishalogen, R³ is hydrogen, R⁴ is alkoxy or —Oaryl, and R⁵ is hydroxyl.

In another embodiment where the N-methanocarba derivative of AMP isaccording to Formula (I), Y is —CH₂— or —CH₂CH₂—, R¹ is NH₂, R² ishalogen, R³ is hydrogen, and R⁴ and R⁵ are both alkoxy or —Oaryl.

In yet another embodiment where the N-methanocarba derivative of AMP isaccording to Formula (I), Y is —CH₂— or —CH₂CH₂—, R¹ is NH₂, R² ishalogen, R³ is hydrogen, and R⁴ and R⁵ form a six membered cyclicstructure —OCH(R)CH₂CH₂O— where R is hydrogen or aryl.

In yet another embodiment where the N-methanocarba derivative of AMP isaccording to Formula (I), the derivative is a triethylamine salt where Yis —CH₂—, R¹ is NH₂, R² is iodo, R³ is hydrogen, R⁴ hydroxyl and R⁵hydroxyl.

In still another embodiment where the N-methanocarba derivative of AMPis according to Formula (I), Q¹ is O; R¹ is N(R⁶)₂ wherein each R⁶ ishydrogen; R² is halogen; R³ is hydrogen; R⁴ is hydroxyl or optionallysubstituted alkoxy; R⁵ is hydroxyl or optionally substituted alkoxy; andY is a linking group linked to the phosphorus atom by a carbon atom.

In another embodiment where the N-methanocarba derivative of AMP isaccording to Formula (I), Q¹ is O; R¹ is N(R⁶)₂ wherein each R⁶ ishydrogen; R² is halogen; R³ is hydrogen; R⁴ is hydroxyl or optionallysubstituted alkoxy; R⁵ is hydroxyl or optionally substituted alkoxy; andY is a C₁-C₆ alkylene.

Other suitable N-methanocarba derivatives of AMP are given in Formula(II) below:

wherein R¹, R², R³, and Y are as previously defined; X is O or S; n is1, 2, or 3; and R⁷ is optionally substituted alkyl or optionallysubstituted aryl, a deuterium enriched isomer thereof, or apharmaceutically acceptable salt thereof.

In one embodiment where the N-methanocarba derivatives of AMP isaccording to Formula (II), Y is —CH₂— or —CH₂CH₂—, R¹ is NH₂, R² ishalogen, R³ is hydrogen, n is 1, X is O, and R⁷ is alkyl or aryl.

In another embodiment where the N-methanocarba derivatives of AMP isaccording to Formula (II), Y is —CH₂— or —CH₂CH₂—, R¹ is NH₂, R² ishalogen, R³ is hydrogen, n is 2, X is S, and R⁷ is methyl.

Still other suitable N-methanocarba derivatives of AMP are given inFormula (III) below:

wherein R¹, R², R³, and Y are as previously defined; and Z is a bond or—O—C(═O)—where the carbonyl carbon is bonded to the oxygen of thebicycle group and the oxygen is bonded to the phosphorus atom, adeuterium enriched isomer thereof, or a pharmaceutically acceptable saltthereof.

In one embodiment where the N-methanocarba derivatives of AMP isaccording to Formula (III), Y is —CH₂— or —CH₂CH₂—, R¹ is NH₂, R² ishalogen, R³ is hydrogen, and Z is a bond or —O—C(═O)—.

Other suitable N-methanocarba derivatives of AMP are given in Formula(IV) below:

wherein R¹, R², R³, R⁵, and Y are as previously defined; R⁸ is hydrogenor optionally substituted alkyl; and R⁸ is optionally substituted alkyl,optionally substituted alkoxy, or optionally substituted aryl, adeuterium enriched isomer thereof, or a pharmaceutically acceptable saltthereof.

In one embodiment where the N-methanocarba derivatives of AMP isaccording to Formula (IV), Y is —CH₂— or —CH₂CH₂—, R¹ is NH₂, R² ishalogen, R³ is hydrogen, R⁵ is hydroxyl, R⁸ is methyl, and R⁹ ismethoxy.

In another embodiment where the N-methanocarba derivatives of AMP isaccording to Formula (IV), Y is —CH₂— or —CH₂CH₂—, R¹ is NH₂, R² ishalogen, R³ is hydrogen, R⁵ is —O-phenyl, R⁸ is methyl, and R⁹ ismethoxy.

Other suitable N-methanocarba derivatives of AMP are given in Formula(V) below:

wherein R¹, R², R³, n, and Y are as previously defined; G is O or S—S;and R¹⁰ is hydrogen, hydroxyl, optionally substituted alkyl, optionallysubstituted alkoxy, or optionally substituted aryl, a deuterium enrichedisomer thereof, or a pharmaceutically acceptable salt thereof.

In one embodiment where the N-methanocarba derivatives of AMP isaccording to Formula (V), Y is —CH₂— or —CH₂CH₂—, R¹ is NH₂, R² ishalogen, R³ is hydrogen, n is 2, G is S—S, and R¹⁰ is hydroxyl.

Still other suitable N-methanocarba derivatives of AMP are given inFormula (VI) below:

wherein R¹, R², R³, and Y are as previously defined; and R¹¹ ishydrogen, optionally substituted alkyl, or optionally substituted aryl.In one embodiment where the N-methanocarba derivatives of AMP isaccording to Formula (VI), Y is —CH₂— or —CH₂CH₂—, R¹ is NH₂, R² ishalogen, R³ is hydrogen, and R¹¹ is alkyl, or aryl, a deuterium enrichedisomer thereof, or a pharmaceutically acceptable salt thereof.

Still other suitable N-methanocarba derivatives of AMP are given inFormula (VII) below:

wherein Q¹ is O or S;

Q² is O or S;

R¹, R², R³, R⁴, and R⁵ are as previously defined above; with the provisothat when Q¹ and Q² are both O, and Formula (VII) is not enriched withdeuterium, then R⁴ and R⁵ are not both hydroxyl; and

Y¹ is a linking group, a deuterium enriched isomer thereof, or apharmaceutically acceptable salt thereof.

The Y¹ linking group is optionally substituted C₁-C₆ alkylene,optionally substituted C₁-C₆ alkenylene, optionally substituted C₁-C₆alkynylene, or optionally substituted —C₁-C₆ alkylene-O—C₁-C₆ alkylene-.In one embodiment, Y¹ is —CH₂—. In another embodiment, Y¹ is —CH₂CH₂—.In yet another embodiment, Y¹ is —CH═CH—.

In another embodiment where the N-methanocarba derivative of AMP isaccording to Formula (VII), Q¹ is O; Q² is S; R¹ is N(R⁶)₂ wherein eachR⁶ is hydrogen; R² is halogen; R³ is hydrogen; R⁴ is hydroxyl oroptionally substituted alkoxy; R⁵ is hydroxyl or optionally substitutedalkoxy; and Y¹ is a C₁-C₆ alkylene.

In another embodiment where the N-methanocarba derivative of AMP isaccording to Formula (VII), Q¹ is S; Q² is O; R¹ is N(R⁶)₂ wherein eachR⁶ is hydrogen; R² is halogen; R³ is hydrogen; R⁴ is hydroxyl oroptionally substituted alkoxy; R⁵ is hydroxyl or optionally substitutedalkoxy; and Y¹ is a C₁-C₆ alkylene.

Specific embodiments of phosphonate or phosphinate N-methanocarbaderivatives of AMP include:

Without being held to theory, it is believed that the spacing of thephosphorus relative to the methanocarba ring is important for in vivoactivity. Among the phosphonate derivatives tested, only the longerspacer of 2 carbons in 11a resulted in effective cardioprotection byprodrug ester derivatives, even though the precursors or unblockednucleotides, i.e. 4 and its higher homologue 9, were active in bothcases. Thus, both the masked diester 11a and its charged precursor 9were clearly protective in vivo. This suggested that enzymaticunblocking of the esters in vivo depended on unhindered steric access tothe phosphonyl group.

The phosphonate or phosphinate N-methanocarba derivatives of AMP have anaffinity for P2X receptors, including cardiac and vascular P2Xreceptors. The P2X receptor affinity can be determined by thedose-response of increases in contractility for the different compounds.Changes in contractility can be measured as changes in sarcomere lengthand Ca²⁺ transients recorded from single isolated myocytes using anepi-fluorescence inverted microscope.

In one embodiment, phosphonate or phosphinate N-methanocarba derivativesof AMP are useful in the treatment of cardiac diseases responsive toactivation of the cardiac P2X receptor such as, for example, cardiachypertrophy and/or cardiac failure resulting from abnormal Ca²⁺homeostasis.

In one embodiment, the compounds disclosed herein are used in thetreatment of cardiac diseases responsive to activation of the cardiacP2X receptor such as cardiomyopathy. Cardiac diseases responsive toactivation of cardiac P2X receptors include, for example, cardiachypertrophy and/or cardiac failure resulting from abnormal Ca²⁺homeostasis. Cardiac hypertrophy is a thickening of the heart muscle(myocardium), which results in a decrease in size of the chamber of theheart, including the left and right ventricles. Alterations in Ca²⁺handling are known to be associated with cardiac hypertrophy. Cardiacfailure is the failure of the heart to maintain a cardiac outputsufficient to meet the metabolic demands of the body. Cardiac failurecan result from any structural or functional cardiac disorder thatimpairs the ability of the heart to fill with or pump a sufficientamount of blood throughout the body. The compounds disclosed herein areparticularly useful in the treatment if cardiac failure resulting fromabnormal Ca²⁺ homeostasis.

The compounds disclosed herein are particularly useful in the treatmentof cardiac diseases responsive to activation of the cardiac P2X receptorassociated with defects in cardiac contractility. Such diseases includemyocardial infarction. As used herein, myocardial infarction, commonlyknown as a heart attack, is a disease state that occurs when the bloodsupply to a part of the heart is interrupted. The resulting ischemia oroxygen shortage causes damage and potential death of heart tissue. Inone embodiment, treatment is done within the within the short-termpost-infarction period. As used herein, the short term post-infarctionperiod is within 48 hours of myocardial infarction. The advantage oftreating in the short term post infarction period is to block thestimulus for cardiac hypertrophy and adverse remodeling at an earlystage of the heart failure progression after myocardial infarction.

In a related embodiment, the compounds have affinity for vascular P2Xreceptors and can be used to treat conditions associated with vascularP2X receptors. Without being held to theory, it is believed thatvascular P2X receptors produce nitric oxide, which can diffuse tomyocytes and improve the function of the myocytes. Thus, by bindingvascular P2X receptors such as endothelial receptors, the phosphonate orphosphinate N-methanocarba derivatives of AMP can be used to treatconditions responsive to an increase in nitric oxide.

Additionally, the compounds disclosed herein are useful for enhancingcardiac function by increasing cardiac muscle contractility and/orincreasing diastolic cardiac muscle relaxation. Included herein are thusmethods of improving cardiac contractile performance in a mammal in needthereof comprising administering a therapeutically effective amount of aphosphonate or phosphinate N-methanocarba derivative of AMP. In oneembodiment, the mammal has had or is suspected of having a myocardialinfarction. In another embodiment, administering is performed within theshort-term post-infarction period.

The compounds disclosed herein are also useful for improving cardiacfunction by administering to a mammal in need of such treatment. As usedherein, improving cardiac function can include, for example, improvingthe ability of the heart to relax, providing favorable remodeling in asubject with heart failure, decreasing fibrosis, decreasing thehypertrophy of cardiac myocytes, and/or improving calcium handling inmyocytes in a heart failure subject.

The compounds disclosed herein can be used to treat systolic ordiastolic heart failure. ‘Systole’ occurs when the heart contracts and‘diastole’ is the relaxation phase of the heart. The increase in −dP/dtor rate of relaxation of the heart muscle in transgenic animalsoverexpressing the P2X₄ receptor suggests that activation of the cardiacP2X receptor can be used to treat diastolic heart failure. Like P2X₄receptor overexpression, treatment with the N-methanocarba derivativesof AMP may be employed for individuals in need of treatment fordiastolic heart failure. Diastolic heart failure is caused when theheart does not fully relax, so it does not fill properly with blood. Byincreasing the rate of relaxation of the heart muscle, theN-methanocarba derivatives of AMP will improve cardiac function inindividuals with diastolic heart failure. Systolic heart failure issometimes referred to as left ventricular failure, and results from adefect or abnormality in the systolic, that is contraction, functionduring the expulsion of blood to the rest of the body. As a result, theamount of blood pumped to the body and to the lungs is reduced, and theventricle, usually enlarges.

In another embodiment, the compounds disclosed herein are used to treatadverse remodeling and injury following ischemia/reperfusion injury. Inone embodiment, the P2X receptor agonists are used to treat individualsin need of treatment for ischemia and reperfusion injury. Ischemia is adeficiency of oxygen in a part of the body causing metabolic changes,usually temporary, which can be due to a constriction or an obstructionin the blood vessel supplying that part. Reperfusion is the restorationof blood flow to an organ or tissue. Ischemia and reperfusion of theheart muscle can cause significant injury with deleterious consequences.Effective therapies that reduce such injury will have significantbenefits in treatment of myocardial infarction and reperfusion injury.

The phosphonate or phosphinate N-methanocarba derivatives of AMP areused to treat a mammal such as a human being.

In one embodiment, the phosphonate or phosphinate N-methanocarbaderivative of AMP is co-administered with an additional agent such as,for example, a beta-adrenergic receptor blocker, an angiotensin receptorblocker or an angiotensin converting enzyme inhibitor or an aldosteronereceptor blocker.

In one embodiment, included herein is a composition comprising aphosphonate or phosphinate N-methanocarba derivative of AMP and apharmaceutically acceptable excipient.

For oral administration, the pharmaceutical preparation can be in liquidform, for example, solutions, syrups or suspensions, or can be presentedas a drug product for reconstitution with water or other suitablevehicle before use. Such liquid preparations can be prepared byconventional means with pharmaceutically acceptable additives such assuspending agents (e.g., sorbitol syrup, cellulose derivatives orhydrogenated edible fats); emulsifying agents (e.g., lecithin oracacia); non-aqueous vehicles (e.g., almond oil, oily esters, orfractionated vegetable oils); and preservatives (e.g., methyl orpropyl-p-hydroxybenzoates or sorbic acid). The pharmaceuticalcompositions can take the form of, for example, tablets or capsulesprepared by conventional means with pharmaceutically acceptableexcipients such as binding agents (e.g., pregelatinized maize starch,polyvinyl pyrrolidone or hydroxypropyl methylcellulose); fillers (e.g.,lactose, microcrystalline cellulose or calcium hydrogen phosphate);lubricants (e.g., magnesium stearate, talc or silica); disintegrants(e.g., potato starch or sodium starch glycolate); or wetting agents(e.g., sodium lauryl sulphate). The tablets can be coated by methodswell-known in the art.

Preparations for oral administration can be suitably formulated to givecontrolled release of the active compound.

For buccal administration, the compositions can take the form of tabletsor lozenges formulated in conventional manner.

For administration by inhalation, the compositions are convenientlydelivered in the form of an aerosol spray presentation from pressurizedpacks or a nebulizer, with the use of a suitable propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol the dosage unit can be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof, e.g., gelatin for use in an inhaler or insufflator can be formulatedcontaining a powder mix of the compound and a suitable powder base suchas lactose or starch.

The compositions can be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion via eitherintravenous, intraperitoneal or subcutaneous injection. Formulations forinjection can be presented in unit dosage form, e.g., in ampoules or inmulti-dose containers, with an added preservative. The compositions cantake such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and can contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient can be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

The compositions can be formulated into creams, lotions, ointments ortinctures, e.g., containing conventional bases, such as hydrocarbons,petrolatum, lanolin, waxes, glycerin, or alcohol. The compositions canalso be formulated in rectal compositions such as suppositories orretention enemas, e.g., containing conventional suppository bases suchas cocoa butter or other glycerides.

In addition to the formulations described previously, the compositionscan also be formulated as a depot preparation. Such long actingformulations can be administered by implantation (e.g., subcutaneouslyor intramuscularly) or by intramuscular injection. Thus, for example,the compositions can be formulated with suitable polymeric orhydrophobic materials (e.g., as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt. Liposomes and emulsions are well known examplesof delivery vehicles or carriers for hydrophilic drugs.

The compositions can, if desired, be presented in a pack or dispenserdevice, which can contain one or more unit dosage forms containing theactive ingredient. The pack can for example comprise metal or plasticfoil, such as a blister pack. The pack or dispenser device can beaccompanied by instructions for administration.

The amount of phosphonate or phosphinate N-methanocarba derivative ofAMP that may be combined with pharmaceutically acceptable excipients toproduce a single dosage form will vary depending upon the host treatedand the particular mode of administration. The specific therapeuticallyeffective amount for a particular patient will depend on a variety offactors including the activity of the specific compound employed, theage, body weight, general health, sex, diet, time of administration,route of administration, rate of excretion, drug combination, and theseverity of the particular disease undergoing therapy. In someinstances, dosage levels below the lower limit of the aforesaid rangemay be more than adequate, while in other cases still larger doses maybe employed without causing any harmful side effects provided that suchhigher dose levels are first divided into several small doses foradministration throughout the day. The concentrations of the compoundsdescribed herein found in therapeutic compositions will vary dependingupon a number of factors, including the dosage of the drug to beadministered, the chemical characteristics (e.g., hydrophobicity) of thecompounds employed, and the route of administration. In general terms,the phosphonate or phosphinate N-methanocarba derivatives of AMP may beprovided in an aqueous physiological buffer solution (for example, 1 cc)containing about 0.2% w/v compound for oral administration. Typical doseranges are about 285 μg/kg of body weight per day in three divideddoses; a preferred dose range is from about 42 μg/kg to about 171 μg/kgof body weight per day. The preferred dosage of drug to be administeredis likely to depend on such variables as the type and extent ofprogression of the disease or disorder, the overall health status of theparticular patient, the relative biological efficacy of the compoundselected, and formulation of the compound excipient, and its route ofadministration, as well as other factors, including bioavailability,which is in turn influenced by several factors. For example, if thecompound is metabolized in the liver or excreted in bile, some of theactive compound absorbed from the gastrointestinal tract will beinactivated by the liver before it can reach the general circulation andbe distributed to its sites of action. It is not believed that thephosphonate or phosphinate N-methanocarba derivatives of AMP will besubject to this first-pass loss. Additionally, because these compoundsare polar and water soluble, it is expected that they will have a smallvolume of distribution, and thus be readily eliminated by the kidney.Moreover, binding of the instant compounds to plasma proteins may limittheir free concentrations in tissues and at their locus of action sinceit is only the unbound drug which equilibriums across the membranereceptor sites. It is anticipated that the phosphate moiety of theinstant compounds may facilitate binding of the compounds to plasmaalbumins, which will in turn influence the amount of free compoundavailable to activate muscle cell P2 purinergic receptors. However, itis expected that such binding to plasma protein will not generally limitrenal tubular secretion of biotransformation since these processes lowerthe free drug concentration and this is rapidly followed by theassociation of this drug-protein complex. Another factor affectingbioavailability is the distribution of the compounds to tissues. Giventhe relatively small size of the compounds and their water solubility,it is anticipated that the compounds will have a relatively fast secondphase of drug distribution. This distribution is determined by both theblood flow to the particular tissue of the organ, such as the heart, aswell as the rate at which the compounds diffuse into the interstitialcompartment from the general circulation through the highly permeablecapillary endothelium (except in the brain). Due to the relativehydrophilicity of these compounds, it is anticipated that there will beno fat or other significant tissue reservoir of the compounds, whichwould account for a third phase of distribution-accumulation.

The invention is further illustrated by the following nonlimitingexamples.

EXAMPLES Example 1 Chemical Synthesis

The phosphonate derivatives on varied carbon skeletons (Table 1) weresynthesized by the methods shown in Schemes 1-6. In some cases, thephosphorous atom was bonded directly to the 5′ carbon atom (Schemes 1,2), while in other cases, a carbon atom was added at that position toform either a saturated or unsaturated nucleotide analogue (Schemes3-5). Alternately, a methylphosphonate group was included in compounds11 and 12, which were otherwise equivalent to 4 and 5, respectively(Scheme 6). The 2 position contained either hydrogen (in compounds 5, 8,10, 12) or Cl (as in the known active compound 3 and the novel analogues4, 7, 9, 11). The reference compound 3 was synthesized by a modificationof the reported method, which lead to improved yields.

Known alcohol 13 was protected as a O-tert-butyldimethylsilyl etherusing TBDPS-Cl, imidazole and DMAP to get compound 14, followed by thereduction of the ethyl ester using DIBAL-H in anhydrous THF resulting incompound 15 in very good yield (Scheme 1). In order to introduce an iodogroup at the 5′ position, a classical two step procedure wasimplemented. This involved the initial activation of the 5′-alcohol as amesylate followed by an S_(N)2 nucleophilic attack of iodide on theactivated 5′-position resulting in the 5′-iodo compound 17 in 95% yield.The iodo compound 17 was subjected to classical Michaelis-Arbuzovreaction conditions with excess triethylphosphite and heating up to 110°C. for 17 h to provide a phosphonate diester 18 in excellent 94% yield(Scheme 1). Desilylation using TBAF resulted in alcohol 19, which was asuitable substrate for Mitsunobu base coupling reactions.

The alcohol 19 was used as a common key intermediate to synthesizephosphonates 4 and 5 (Scheme 2). Synthetic procedures for phosphonates 4and 5 involved initial Mitsunobu base coupling reaction usingtriphenylphosphine, diisopropyl azodicarboxylate, and the correspondingpurine base followed by amination at the 6 position of the purine ringusing 2M NH₃ in isopropanol (Scheme 2). Finally, the simultaneousdeprotection of both the phosphonate diester and the acetonide of 21 and23 was achieved upon treatment with freshly opened iodotrimethylsilaneto get target phosphonates 4 and 5, respectively (Scheme 2). Efforts touse alternative relatively milder reagents, bromotrimethylsilane andDowex-50 ion exchange resin, resulted in partial deprotection (resultsnot shown).

The synthetic routes to the elongated saturated and unsaturatedphosphonate derivatives 7-10 are shown in Schemes 3-5. The saturatedphosphonate 10 was synthesized by oxidation of known alcohol 24 to the5′-aldehyde 25 in 80% yield. It is noteworthy that not even a smallamount of decomposition of the aldehyde was observed after storage atroom temperature (rt) for several days. The α,β-unsaturated alkylphosphonate ester 26 was prepared from aldehyde 25 in a Wittig-typereaction using tetraisopropyl methylenediphosphonate and sodium hydridein anhydrous THF. The E-configuration of the resulting alkene could beinferred from the large coupling constant (³J=17.1 Hz). Aminationfollowed by hydrolysis of phosphonate diester and acetonide resulted inα,β-unsaturated alkyl phosphonate 27 (Scheme 3).

Catalytic hydrogenation of 27 in the presence of H₂ (3 bar), palladiumon carbon and MeOH:2M aq. NaOH (1:1, v/v) resulted in the expectedolefin reduction and dechlorination to give the corresponding saturatedphosphonate diester 28. Compound 28 was converted to the long chainsaturated alkyl phosphonate 10 using the previously describediodotrimethylsilane deprotection reaction conditions. To our surprise,our various efforts to synthesize 36 (Scheme 5) from 27 by olefinreduction, running the reaction at atmospheric pressure and using lessweight percent of catalyst, either resulted in an incomplete reaction orgenerated an inseparable mixture of dehalogenated and halogenatedproducts (results not shown).

Hence, in order to synthesize phosphonates 8 and 9, we decided toinstall the phosphonate diester before the Mitsunobu base couplingreaction, as described in Schemes 4 and 5. The 5′-alcohol of compound 15was oxidized using Dess-Martin periodinane reaction to get aldehyde 29.Similar to the aldehyde 25, aldehyde 29 also displayed considerablestability at rt. The α,β-unsaturated alkyl phosphonate diester 30 wasobtained using the previously described Wittig-type reaction conditions.Desilylation under standard conditions resulted in the formation ofalcohol 31, which served as the key intermediate for the synthesis oflong chain unsaturated and saturated alkyl phosphonates 8 and 9,respectively.

A Mitsunobu base coupling reaction on compound 31 usingtriphenylphosphine, 6-chloropurine, and diisopropyl azodicarboxylatefollowed by amination and hydrolysis of the phosphonate diester andacetonide resulted in formation of long chain α,β-unsaturated alkylphosphonate 8. Sequential catalytic hydrogenation of the resulting vinylphosphonate diester 31 in the presence of palladium on carbon, aMitsunobu base coupling reaction, and amination provided the long chainsaturated phosphonate diester 36. Simultaneous deprotection of thephosphonate diester and the acetonide using iodotrimethylsilane resultedin the desired phosphonate 9 along with the formation of correspondingdehalogenated product to yield phosphonate 10.

The synthetic approach to methylphosphonates 11 and 12 is shown inScheme 6. It involved initial 5′-bromination using CBr₄ and treatmentwith triphenylphosphine and triethylamine to result in 5′-bromosugar 37in 81% yield. A subsequent Michaelis-Arbuzov reaction using diethylmethylphosphite, followed by desilylation with TBAF gave the5′-methylphosphonate monoester 38 with 1-alcohol 39 as an inseparablemixture of diastereomers. A further Mitsunobu base coupling reactionwith 2,6-dichloropurine, followed by amination and final deprotectiongave the desired methylphosphonate 11 along with correspondingdehalogenated methylphosphonate 12.

Example 3 Biological Evaluation

Various phosphonate derivatives were infused subcutaneously individuallyvia an Alzet minipump in CSQ mice. After 28 days of infusion, the invivo heart function was assessed using echocardiography-derivedfractional shortening (FS), which is the ratio of the change in thediameter of the left ventricle between the contracted and relaxedstates. Thus, a lower percentage represents a decrease in function. Twoof the phosphonates, 4 and 9, were able to cause an improved FS ascompared to vehicle (FIG. 1, Table 1) and in comparison to the referencenucleotide 3. Other analogues tested in this model (2-H analogues 5, 8,and 10, and 2-Cl analogues 7 and 11) had lower FS values. Compounds 7,8, and 10 were not protective at this dose, i.e., FS in vehiclecontrol-treated CSQ mice was similar to that from mice treatedchronically with these nucleotides. Thus, in the saturated phosphonateseries, the orientation of the phosphorous relative to the methanocarbaring was somewhat structurally permissive, although inclusion of anolefin in the spacer prevented the cardioprotective action. In 11, oneOH group of the phosphonate has been replaced with CH₃. This reduces theoverall charge on the molecule and is intended to make it morebioavailable. Evidently, the binding site of the receptor requires bothoxygens for the most favorable improvement in FS. Therefore, assummarized in FIG. 1, the most significant improvement in FS wasassociated with the saturated homologues containing a 2-Cl substitutionand an unmodified phosphonate group, 4 and 9.

An echocardiographic image of compound 4-versus normal saline(NS)-infused CSQ hearts is shown in FIG. 2. An increased shortening ofboth the septum and left ventricular (LV) free wall was evident in theheart from mice treated with 4 in comparison to that from vehicle(NS)-infused mice.

The ability of the nucleotide analogues to activate the human P2Y₁receptor (Table 1) was also investigated. This receptor is vasodilatory,and agonist action at this subtype would be expected to be relevant tothe observed cardiac effects. Compound 3 was previously reported toactivate phospholipase C (PLC) mediated by the human P2Y₁ receptor. Thephosphonate derivatives were tested in a FLIPR assay of calcium fluxinduced in 1321N1 astrocytoma cells stably expressing the human P2Y₁receptor. The known P2Y₁ receptor agonist 2-MeSADP induced a Ca²⁺ fluxwith an EC₅₀ of 10.3±0.4 nM (n=3) in the transfected cells (FIG. 3), butin control 1321N1 astrocytoma cells there was no change in intracellularCa²⁺ in response to 10 μM 2-MeSADP. At concentrations up to 10 μM, thephosphonate analogues 4-11 produced no effect in the same assay.However, compound 3 was active in this assay as an agonist, with an EC₅₀of 722±55 nM (n=3). The maximal effect of 3 was about 80% of that of thefull agonist 2-MeSADP.

In conclusion, the range of carbocyclic nucleotide analogues thatrepresent potential candidates for the treatment of heart failure hasbeen expanded. A more chemically and biologically stable linkage thanthe phosphate group in compound 3 has been introduced in the form ofphosphonate groups, which in several cases preserve heart contractilefunction in a genetic model of heart failure. Facile routes for thesynthesis of phosphonate analogues of compound 3 in the conformationallyconstrained (N)-methanocarba series were developed usingMichaelis-Arbuzov and Wittig reactions. A further advantage of thephosphonate linkage is that the undesired activity as agonist of theP2Y₁ receptor has been eliminated. The beneficial effects of thesenucleotidase-resistant agonists can now be explored in additional modelsof cardiac failure and cardiomyopathy.

Example 3 Additional Chemical Synthesis

The novel, charged 5′-phosphonate (4a-7a, Table 2a) and 5′-esterderivatives (8a-17a, Table 2) in the (N)-methanocarba series weresynthesized by the methods shown in Schemes 1a-4a. Modifications ofcompound 3 include S substitution of O at phosphorus (4a, 5a) (Scheme1a) and the introduction of deuterium at the 5′ position in 6a (Scheme2a), both of which are believed to improve stability in vivo. Compound7a was prepared as the 2-iodo equivalent of 4 (Scheme 3a). Thecorresponding ester-masked derivatives (8a-17a), including prodrugderivatives of the reference compounds 3, 4, and 7, were prepared,either from intermediates or as shown in Schemes 1a-4a. Most of theesters consisted of diethyl esters, but one diisopropyl ester 11a wasincluded. Phosphotriester derivatives included 8a (and its dideuteratedequivalent 15a) and thio derivatives 12a-14a. In some cases, thephosphorus atom of a phosphonate was bonded directly to the 5′ carbonatom, e.g. in 2-iodo derivative 16a, while in other cases, a carbon atomwas added at that position to form an extended saturated phosphonateanalogue, e.g. in diethylester derivative 10a. Compound 17a was preparedas the 2-ethynyl equivalent of 9a.

The synthetic route for various thio derivatives is shown in Scheme 1a.Previously reported nucleoside 18a used as key intermediate to generatethese thio analogues, initial acetonide deprotection using 10% aqueoustrifluoroacetic acid in CH₂Cl₂ afforded the 2′,3′ and 5′-trihydroxynucleoside 19a. 5′-Thiophosphate 5a could be generated from intermediate19a by treating it with thiophosphoryl chloride,1,8-bis-(dimethylamino)naphthalene (proton sponge) and pyridine followedby quenching the reaction with tetraethylammonium bicarbonate (TEAB).However, we anticipate that subjecting nucleoside 19a to the sameconditions followed by quenching with EtOH should provide5′-thiophosphate-di-ethylester 13a. 5′-Iodination of the nucleosideintermediate 18a using PPh₃, I₂, and imidazole in anhydrous THF affordedthe 5′-iodo nucleoside 29a. Subsequent deprotection of the acetonideprotecting group of 29a using Dowex-50 resin afforded the5′-iodo-2′,3′-dihydroxy nucleoside 21a (Scheme 1a) as a key intermediateto generate 5′-phosphorothiate 12a, 5′-phosphorothiate diethylester 4aand 5′-phosphorodithioate diethylester 14a (Scheme 1a). Treating keynucleotide 21a with sodium O,O-diethylthiophosphate in EtOH couldgenerate 5′-phosphorothiate diethylester 4a. Treatment of nucleoside 21awith trisodium thiophosphate in H₂O for 3 d afforded 5′-phosphorothiate12a in 57% yield. Alternately, reacting the nucleotide 21a with thediethyl dithiophosphate potassium salt in DMF would yield5′-phosphorodithioate diethylester 14a (Scheme 1a).

5′-Dideuteromethyl (N)-methanocarba monophosphate 6a and its diethylester analogue 15a were synthesized from nucleoside 22a (Scheme 2a).Initial amination at the C6 position of the purine base using 2MNH₃/i-ProH followed by reduction of the ethyl ester using LiBD₄ inanhydrous THF provided the 5′-hydroxyduetiromethyl nucleoside 24a. Thenucleoside 24a was phosphorylated by first reacting with eitherdi-t-butyl or di-ethyl analogue of N,N-diethylphosphoramidite andtetrazole followed by treatment with m-chloroperbenzoic acid to affordthe corresponding di-t-butyl or di-ethyl phosphate diester derivatives25a and 26a, respectively. Both t-butyl groups and the acetonide groupin 26a were deprotected simultaneously by using Dowex-50 resin in theacid form to afford the 5′-dideuteromethyl monophosphate 6a. Thesynthesis of dideuteromethyl phosphonate diethylester 15a could beachieved by chemoselectively deprotection of acetonide group ofnucleotide 25a using Dowex-50 resin (Scheme 2a).

Synthetic procedures for various 2-position substituted adeninephosphonates (7a, 32a) and phosphonate diethylesters (9a, 16a, 17a)started with installation of the nucleobase on a previously describedsugar 27a by a Mitsunobu base coupling reaction using PPh₃, diisopropylazodicarboxylate, and 6-chloro-2-iodopurine to generate6-chloro-2-iodo-purine nucleotide 28a. Next, amination at the C6position of compound 28a using 2M NH₃ in isopropanol (Scheme 3a)afforded the nucleotide 29a. Finally, simultaneous deprotection of boththe phosphonate diester and acetonide of 29a was achieved upon treatmentwith freshly opened iodotrimethylsilane in CH₂Cl₂ to obtain targetphosphonate 7a in 49% yield. Alternately, treatment of 2-chloro (30a)and 2-iodo (29a) nucleotides with Dowex-50 ion-exchange resin resultedin a chemo-selective deprotection of acetonide and resulted in aformation of corresponding 2-chloro and 2-iodo diethyl phosphonateesters 9a and 16a, respectively. 2-Ethynyl-substituted phosphonatediethylester 17a and phosphonate 32a derivatives were generated usinginitial installation of acetylene group following the classicalSonogashira coupling protocol using trimethylsilylacetylene, Pd(Ph₃)₄,CuI, TEA in anhydrous DMF. Subsequently, TMS protection at acetylene wasremoved using TBAF in anhydrous THF to afford 2-ethynyl-substitutedphosphonate diester 31a. Similar to the other derivatives,2-ethynyl-substituted adenine phosphonates (32a) and phosphonatediethylesters (17a) were synthesized using the Dowex-basedchemoselective and iodotrimethylsilylane-based full deprotectionprotocols, respectively.

The saturated long-chain phosphonate esters 10a and 11a were synthesizedby oxidation of known alcohol 33a to the 5′-aldehyde 34a in 80% yield.It is noteworthy that not even a small amount of decomposition of thealdehyde was observed after storage at room temperature (rt) for severald. The α,β-unsaturated alkyl phosphonate ester 35a could be preparedfrom aldehyde 34a in a Wittig-type reaction using tetraethylmethylenediphosphonate and sodium hydride in anhydrous THF. Amination atthe 6-position of purine base would give us compound 36a, which could besubjected to chemo selective reduction of alkene using the diimidegenerated in situ from O-nitrobenzenesulfonylhydrazide, Et₃N in CH₂Cl₂to get the long-chain saturated phosphonate diethyl ester 37a. Acetonidedeprotection of compounds 37a and 38a using Dowex-50 resin resulted information of diethyl and diisopropyl phosphonate esters, targetcompounds 10a and 11a, respectively.

Example 4 Additional Biological Evaluation

Various phosphonate derivatives were infused subcutaneously individuallyvia an Alzet minipump in CSQ mice. After 14 days of infusion, the invivo heart function was assessed using echocardiography-derivedfractional shortening (FS), which is the ratio of the change in thediameter of the left ventricle between the contracted and relaxed states(Table 2). Thus, a lower percentage represents a decrease in function.

The structure activity relationships (SARs) of the charged nucleotideanalogs were explored. Two-week infusion of the 2-iodo phosphonatederivative 7a (n=5 mice) did not improve FS or prevent LV wall thinningin mice with heart failure (data not shown). Thus, 2-Cl substitution ofthe adenine moiety as in phosphonate 4 was essential for activity;substitution with iodo in 7a abolished protection. Several new phosphateand phosphonate analogues, such as thio derivatives, were compared. A2-week infusion of thiophosphate 4a (n=5), containing a 5′-thioester,could protect the CSQ mice with a better preservation of LV septal(0.492±0.012 mm) and posterior (0.493±0.016 mm) wall thickness ascompared those obtained in NS-infused (both septal and posterior:0.450±0.007 mm) CSQ mice (P<0.05, data not shown). Thus, substitution ofoxygen with sulfur was tolerated.

The structure activity relationships (SARs) of the masked (uncharged)nucleotide analogs was explored using the same experimental model. Onlysome of the ester derivatives of the previously characterizedcardioprotective agents 3, 4, and 9 were shown to act in vivo. Thesefindings implied that a cleavage step in vivo to liberate the chargednucleotide active drug was necessary. Among the prodrug derivatives,diisopropyl ester 11a of phosphonate(1′S,2′R,3′S,4′R,5′S)-4′-(6-amino-2-chloropurin-9-yl)-2′,3′-(dihydroxy)-1′-(phosphonoethylene)-bicyclo[3.1.0]hexane9 was highly efficacious. This phosphonate diester resulted in animproved FS as compared to vehicle (FIG. 4, Table 2). In mice infusedwith compound 11a, the LV posterior wall thickness and septal thicknessduring systole and the LV posterior wall thickness during diastole weregreater than those in NS-infused CSQ mice (FIG. 5). Other analoguestested in this model, e.g. 7a and 9a, had lower FS values and were notprotective at this dose, i.e., FS in CSQ mice infused with compounds 7aand 9a was similar to that from normal saline-infused control mice.Furthermore, two-week infusion of 7 (n=5 mice) or 9a (n=4) did notimprove FS or prevent LV wall thinning in CSQ mice with heart failure(data not shown). Thus, the lower homologue 1′-(phosphonoethylene)derivative 9a was less active than 11a, suggesting that unblocking ofthe esters in vivo depended on unhindered steric access to thephosphonyl group.

Experimental Procedures for Example 1 General Methods:

Compound 13 was either synthesized as reported or obtained as a customsynthesis from Natland International Corporation (Research TrianglePark, N.C.). All other reagents and solvents (regular and anhydrous)were of analytical grade and obtained from commercial suppliers and usedwithout further purification. Reactions were conducted under anatmosphere of argon whenever anhydrous solvents were used. All reactionswere monitored by thin-layer chromatography (TLC) using silica gelcoated plates with a fluorescence indicator which were visualized: a)under UV light, b) by dipping in 5% conc. H₂SO₄ in absolute ethanol(v/v) followed by heating, or c) by dipping in a solution ofanisaldehyde:H₂SO₄ (1:2, v/v) in MeOH followed by heating. Silica gelcolumn chromatography was performed with silica gel (SiO₂, 200-400 mesh,60 Å) using moderate air pressure. Evaporation of solvents was carriedout under reduced pressure at a temperature below 50° C. After columnchromatography, appropriate fractions were pooled, evaporated and driedat high vacuum for at least 12 h to give the obtained products in highpurity. ¹H NMR and ³¹P NMR ascertained sample purity. No corrections inyields were made for solvent of crystallization. ¹H NMR and ³¹P NMRspectra were recorded at 300 MHz and 121.5 MHz, respectively. Chemicalshifts are reported in parts per million (ppm) relative totetramethylsilane or deuterated solvent as the internal standard (dH:CDCl₃ 7.26 ppm). For compounds 38-41, the integral of the H3′-signal ofthe least predominant isomer was set to 1.0. Systematic compound namesfor bicyclic nucleosides are given according to the von Baeyernomenclature. High resolution mass spectroscopic (HRMS) measurementswere performed on a proteomics optimized Q-TOF-2 (Micromass-Waters)using external calibration with polyalanine. Observed mass accuraciesare those expected on the basis of known performance of the instrumentas well as the trends in masses of standard compounds observed atintervals during the series of measurements. Reported masses areobserved masses uncorrected for this time-dependent drift in massaccuracy.

Purification of the nucleotide derivatives for biological testing wasperformed by HPLC with a Luna 5 μm RP-C18(2) semipreparative column(250×10.0 mm; Phenomenex, Torrance, Calif.) under the followingconditions: flow rate of 2 mL/min; 10 mM triethylammonium acetate(TEAA)-CH₃CN from 100:0 (v/v) to 70:30 (v/v) in 30 min and isolated inthe triethylammonium salt form. Analytical purity of compounds waschecked using a Hewlett-Packard 1100 HPLC equipped with Zorbax SB-Aq 5μm analytical column (50×4.6 mm; Agilent Technologies Inc, Palo Alto,Calif.). Mobile phase: linear gradient solvent system: 5 mM TBAP(tetrabutylammonium dihydrogenphosphate)-CH₃CN from 80:20 to 40:60 in 13min; the flow rate was 0.5 mL/min. Peaks were detected by UV absorptionwith a diode array detector at 254, 275, and 280 nm. All derivativestested for biological activity showed >99% purity by HPLC analysis(detection at 254 nm).

Ethyl-(1S,2R,3S,4S,5S)-2,3-O-(isopropylidene)-4-O-(tert-butyldimethylsilyl)-bicyclo[3.1.0]hexanecarboxylate(14): Known alcohol 13 (0.83 g, 3.40 mmol) was coevaporated withanhydrous toluene (2×10 mL) and dissolved in anhydrous CH₂Cl₂ (25 mL).Imidazole (0.69 g, 10.20 mmol), DMAP (0.04 g, 0.34 mmol) andtert-butylchlorodiphenylsilane (1.74 mL, 6.81 mmol) were added. Afterstirring at rt for 16 h, the reaction mixture was diluted with CH₂Cl₂(50 mL) and washed with sat. aq. NaHCO₃ (1×30 mL). The aqueous phase wasback-extracted with CH₂Cl₂ (2×50 mL). The combined organic phase wasevaporated to dryness, and the resulting crude residue was purified bysilica gel column chromatography (0-8% EtOAc in petroleum ether, v/v) toafford compound 14 (1.52 mg, 93%) as a colorless oil. R_(f)=0.3 (10%EtOAc in CH₂Cl₂, v/v); ESI-HRMS m/z 519.1981 ([M+K]⁺, C₂₈H₃₆O₅Si.K⁺:Calcd. 519.1969); ¹H NMR (CDCl₃) δ 7.69-7.80 (m, 4H, Ph), 7.32-7.45 (m,6H, Ph), 5.11 (d, 1H, J=6.6 Hz), 4.42 (t, 1H, J=6.1 Hz), 3.99-4.18 (m,3H), 2.17-2.25 (m, 1H), 1.91 (t, 1H, J=5.5 Hz), 1.57 (s, 3H), 1.45-1.53(m, 1H), 1.18-1.24 (m, 6H), 1.07 (s, 9H).

(1S,2R,3S,4S,5S)-1-Hydroxymethyl-2,3-O-(isopropylidene)-4-O-(tert-butyldimethylsilyl)-bicyclo[3.1.0]hexane (15): Compound 14 (0.98 g, 2.04 mmol) was coevaporatedwith anhydrous toluene (2×20 mL), dissolved in anhydrous THF (30 mL) andcooled to −70° C. DIBAL-H (1.5 M in toluene 10.8 mL, 16.32 mmol) wasadded slowly to this solution over 20 min. After stirring at −70° C. for3 h, the reaction was quenched with the very careful addition ofice-cold MeOH (20 mL), followed by warming the reaction mixture to rt. 1M cold H₂SO₄ (20 mL) was added to the mixture and it was stirred for 1h, followed by addition of CH₂Cl₂ (100 mL). The phases were separated,and the aqueous phase was extracted with CH₂Cl₂ (3×35 mL). The combinedorganic phase was evaporated to dryness, and the resulting residue waspurified by silica gel column chromatography (0-45% EtOAc in petroleumether, v/v) to afford compound 15 (0.74 g, 82%) as a colorless oil.R_(f)=0.4 (50% EtOAc in petroleum ether, v/v); ESI-HRMS m/z 461.2112([M+Na]⁺, C₂₆H₃₄O₄Si.Na⁺: Calcd. 461.2124); ¹H NMR (CDCl₃) δ7.71-7.78(m, 4H, Ph), 7.31-7.44 (m, 6H, Ph), 4.73 (d, 1H, J=6.5 Hz), 4.44 (t, 1H,J=6.5 Hz), 4.09 (t, 1H, J=6.5 Hz), 3.59-3.67 (m, 1H), 3.41-3.49 (m, 1H),1.57-1.59 (m, 4H), 1.33 (t, 1H, J=5.5 Hz), 1.20 (s, 3H), 1.07 (s, 9H),0.56-0.68 (m, 1H).

(1S,2R,3S,4S,5S)-2,3-O-(Isopropylidene)-1-methanesulfonyloxymethyl-4-O-(tert-butyldimethylsilyl)-bicyclo[3.1.0]hexane(16): Compound 15 (0.59 g, 1.36 mmol) was coevaporated with anhydroustoluene (2×20 mL), dissolved in anhydrous CH₂Cl₂ (30 mL) and cooled to0° C. Triethylamine (0.95 mL, 6.79 mmol) and methanesulfonyl chloride(0.22 mL, 2.72 mol) were added at 0° C. over 10 min. After warming thereaction mixture to rt, it was stirred for 17 h. Then, ice-cold H₂O (25mL) was added and the mixture was extracted with EtOAc (2×45 mL). Thecombined organic phase was washed with sat. aq. NaHCO₃ (2×35 mL) andevaporated to dryness. The resulting residue was purified by silica gelcolumn chromatography (0-50% EtOAc in petroleum ether, v/v) to affordcompound 16 (0.68 g, 96%) as a colorless oil. R_(f)=0.5 (50% EtOAc inpetroleum ether, v/v); ESI-HRMS m/z 555.1623 ([M+K]⁺, C₂₇H₃₆O₆SSi.K⁺:Calcd. 555.1639); ¹H NMR (CDCl₃) δ 7.69-7.77 (m, 4H, Ph), 7.31-7.45 (m,6H, Ph), 4.69 (d, 1H, J=6.5 Hz), 4.47 (t, 1H, J=6.5 Hz), 4.37-4.43 (dd,1H, J=10.9 Hz), 4.07 (t, 1H, J=6.5 Hz), 3.90-3.95 (dd, 1H, J=10.9 Hz),2.99 (s, 3H), 1.67-1.72 (m, 2H), 1.55 (s, 3H), 1.20 (s, 3H), 1.08 (s,9H), 0.72-0.79 (m, 1H).

(1S,2R,3S,4S,5S)-1-Iodomethyl-2,3-O-(isopropylidene)-4-O-(tert-butyldimethylsilyl)-bicyclo[3.1.0]hexane(17): Compound 16 (0.68 g, 1.32 mmol) was coevaporated with anhydroustoluene (2×20 mL), and the residue dissolved in anhydrous 1,4-dioxane(25 mL). NaI (0.59 g, 3.94 mol) was added to the mixture, and it washeated to 65° C. After stirring for 17 h, the reaction mixture wascooled to rt and diluted with H₂O (25 mL) and CH₂Cl₂ (75 mL). The phaseswere separated, and the aqueous phase was extracted with CH₂Cl₂ (3×35mL). The combined organic phase was evaporated to dryness, and theresulting residue was purified by silica gel column chromatography(0-20% EtOAc in petroleum ether, v/v) to afford compound 17 (0.69 g,95%) as a colorless oil. R_(f)=0.5 (20% EtOAc in petroleum ether, v/v);ESI-HRMS m/z 549.1322 ([M+H]⁺, C₂₆H₃₃IO₃Si.H⁺: Calcd. 549.1322); ¹H NMR(CDCl₃) δ 7.68-7.77 (m, 4H, Ph), 7.32-7.46 (m, 6H, Ph), 4.69 (d, 1H,J=6.5 Hz), 4.43 (t, 1H, J=6.5 Hz), 4.09 (t, 1H, J=6.5 Hz), 3.55-3.60(dd, 1H, J=10.5 Hz), 3.97-4.02 (dd, 1H, J=10.5 Hz), 2.02 (t, 1H, J=4.9Hz), 1.54-1.57 (s, 4H), 1.20 (s, 3H), 1.07 (s, 9H), 0.83-0.90 (m, 1H).

Diethyl-(1S,2R,3S,4S,5S)-2,3-O-(isopropylidene)-4-O-(tert-butyldimethylsilyl)-bicyclo[3.1.0]hexanephosphonate (18): Compound 17 (0.68 g, 1.23 mmol) was dissolved intriethylphosphite (17 mL), and the mixture was heated to 110° C. Afterstirring for 17 h, the reaction mixture was cooled to rt and evaporatedto dryness. The resulting residue was purified by silica gel columnchromatography (0-90% EtOAc in petroleum ether, v/v) to afford compound18 (0.65 g, 94%) as a colorless oil. R_(f)=0.3 (EtOAc); ESI-HRMS m/z559.2665 ([M+H]⁺, C₃₀H₄₃O₆PSi.H⁺: Calcd. 559.2645); ¹H NMR (CDCl₃) δ7.71-7.77 (m, 4H, Ph), 7.30-7.43 (m, 6H, Ph), 4.80 (d, 1H, J=6.5 Hz),4.47 (t, 1H, J=6.5 Hz), 4.10 (t, 1H, J=6.5 Hz), 3.91-4.05 (m, 4H), 2.22(t, 1H, J=16.5 Hz), 1.63-1.71 (m, 1H), 1.57-1.61 (m, 2H), 1.55 (s, 3H),1.22 (t, 3H, J=7.2 Hz), 1.20 (t, 3H, J=7.2 Hz), 1.19 (s, 3H), 1.07 (s,9H), 0.53-0.60 (m, 1H). ³¹P NMR (CDCl₃) δ29.93.

Diethyl-(1S,2R,3S,4S,5S)-4-hydroxy-2,3-O-(isopropylidene)-bicyclo[3.1.0]hexane phosphonate (19): Compound 18 (0.65 g, 1.16 mmol) wasdissolved in a mixture of THF (20 mL) and tetrabutylammonium fluoride (1M in THF, 2.91 mL, 2.91 mmol). After stirring for 17 h, the reactionmixture was evaporated to dryness. The resulting residue was purified bysilica gel column chromatography (0-7% MeOH in EtOAc, v/v) to affordcompound 19 (0.33 g, 88%) as a colorless oil. R_(f)=0.3 (5% MeOH inEtOAc, v/v); ESI-HRMS m/z 321.1466 [M+H]⁺, C₁₄H₂₅O₆P.H⁺: Calcd.321.1467); ¹H NMR (CDCl₃) δ 5.02 (d, 1H, J=6.1 Hz), 4.50-4.58 (m, 2H),4.02-4.17 (m, 4H), 2.32-2.37 (m, 1H), 2.26 (t, 1H, J=16.5 Hz), 1.88-1.96(m, 1H), 1.61-1.74 (m, 1H), 1.54 (s, 3H), 1.32 (t, 6H, J=7.2 Hz), 1.28(s, 3H), 1.21-1.27 (m, 1H), 0.60-0.67 (m, 1H). ³¹P NMR (CDCl₃) δ29.01.

Diethyl-(1′S,2′R,3′S,4′R,5′S)-4′-(2,6-dichloropurin-9-yl)-2′,3′-O-(isopropylidene)-bicyclo[3.1.0]hexanephosphonate (20): Diisopropyl azodicarboxylate (97 μL, 0.49 mmol) wasadded at rt to a mixture of triphenylphosphine (128 mg, 0.49 mmol) and2,6-dichloropurine (92 mg, 0.49 mmol) in anhydrous THF (3 mL). Afterstirring for 30 min, a solution of compound 19 (78 mg, 0.25 mmol) in THF(3 mL) was added to the mixture. After stirring for 51 h, the reactionmixture was evaporated to dryness. The resulting residue was purified bysilica gel column chromatography (0-4% MeOH in EtOAc, v/v) to affordnucleoside 20 (90 mg, 75%) as a white solid material. R_(f)=0.5 (5% MeOHin EtOAc, v/v); ESI-HRMS m/z 491.1013 [M+H]⁺, C₁₉H₂₅Cl₂N₄O₅P.H⁺: Calcd.491.1018); ¹H NMR (CDCl₃) δ 8.82 (s, 1H), 5.39 (d, 1H, J=6.5 Hz), 5.10(s, 1H), 4.61 (d, 1H, J=6.5 Hz), 4.02-4.21 (m, 4H), 2.46 (t, 1H, J=16.5Hz), 1.91-2.06 (m, 1H), 1.74-1.82 (m, 1H), 1.54 (s, 3H), 1.32 (t, 3H,J=7.2 Hz), 1.26 (t, 3H, J=7.2 Hz), 1.24 (s, 3H), 1.08-1.21 (m, 1H),0.97-1.06 (m, 1H).

Diethyl-(1′S,2′R,3′S,4′R,5′S)-4′-(6-amino-2-chloropurin-9-yl)-2′,3′-O-(isopropylidene)-bicyclo[3.1.0]hexanephosphonate (21): Nucleoside 20 (90 mg, 0.19 mmol) was treated with 2 MNH₃ in i-PrOH (5 mL), and the mixture was heated to 70° C. and stirredfor 17 h. The reaction mixture was evaporated to dryness, and theresulting residue was purified by silica gel column chromatography (0-5%MeOH in CH₂Cl₂, v/v) to afford nucleoside 21 (70 mg, 80%) as a whitesolid material. R_(f)=0.5 (5% MeOH in CH₂Cl₂, v/v); ESI-HRMS m/z472.1519 [M+H]⁺, C₁₉H₂₇ClN₅O₅P.H⁺: Calcd. 472.1517); ¹H NMR (CDCl₃) δ8.31 (s, 1H), 5.98 (s, 2H), 5.36 (d, 1H, J=7.1 Hz), 4.97 (s, 1H), 4.61(d, 1H, J=6.5 Hz), 4.03-4.19 (m, 4H), 2.39 (t, 1H, J=16.5 Hz), 2.03-2.17(m, 1H), 1.70-1.77 (m, 1H), 1.52 (s, 3H), 1.32 (t, 3H, J=7.2 Hz), 1.25(t, 3H, J=7.2 Hz), 1.23 (s, 3H), 1.18-1.21 (m, 1H), 0.96-1.04 (m, 1H).

(1′S,2′R,3′S,4′R,5′S)-4′-(6-Amino-2-chloropurin-9-yl)-2′,3′-(dihydroxy)-1′-(phosphonomethylene)-bicyclo[3.1.0]hexane(4): Nucleoside 21 (30 mg, 0.064 mmol) was coevaporated with anhydroustoluene (3×3 mL) and dissolved in anhydrous CH₂Cl₂ (3 mL). To thissolution was added iodotrimethylsilane (91 μl, 0.64 mmol). Afterstirring for 17 h, the reaction mixture was cooled to 0° C. followed bythe addition of ice-cold H₂O (25 mL) and CH₂Cl₂ (25 mL). The phases wereseparated, and the aqueous phase washed with CH₂Cl₂ (1×35 mL) anddiethyl ether (3×35 mL). The resulting aqueous phase evaporated todryness and purified by HPLC (retention time: 19.1 min) to afford 4 (8.5mg, 23%) as a white solid material. ESI-HRMS m/z 374.0397 [M−H]⁻,C₁₂H₁₄ClN₅O₅P⁻: Calcd. 374.0421); ¹H NMR (D₂O) δ8.21 (s, 1H), 4.71 (s,1H), 4.57 (d, 1H, J=6.6 Hz), 4.01 (d, 1H, J=6.6 Hz), 3.19 (q, 24H, J=7.2Hz), 2.23 (t, 1H, 15.5 Hz), 1.63-1.77 (m, 2H), 1.42-1.49 (m, 1H), 1.26(t, 36H), 0.96-1.04 (m, 1H). ³¹P NMR (D₂O) δ 23.68. Purity >99% by HPLC(retention time: 4.51 min).

Diethyl-(1′S,2′R,3′S,4′R,5′S)-4′-(6-chloropurin-9-yl)-2′,3′-O-(isopropylidene)-bicyclo[3.1.0]hexanephosphonate (22): Diisopropyl azodicarboxylate (100 μL, 0.50 mmol) wasadded at rt to a mixture of triphenylphosphine (133 mg, 0.50 mmol) and6-chloropurine (96 mg, 0.50 mmol) in anhydrous THF (3 mL). Afterstirring the mixture for 30 min, a solution of compound 19 (81 mg, 0.26mmol) in THF (3 mL) was added. After stirring for 17 h, the reactionmixture was evaporated to dryness. The resulting residue was purified bysilica gel column chromatography (0-4% MeOH in EtOAc, v/v) to affordnucleoside 22 (100 mg, 87%) as a white solid material. R_(f)=0.5 (5%MeOH in EtOAc, v/v); ESI-HRMS m/z 457.1417 [M+H]⁺, C₁₉H₂₆ClN₄O₅P.H⁺:Calcd. 457.1408); ¹H NMR (CDCl₃) δ 8.84 (s, 1H), 8.78 (s, 1H), 5.39 (d,1H, J=6.5 Hz), 5.15 (s, 1H), 4.62 (d, 1H, J=6.5 Hz), 4.07-4.21 (m, 4H),2.44 (t, 1H, J=16.5 Hz), 1.94-2.18 (m, 1H), 1.83-1.90 (m, 1H), 1.58 (s,3H), 1.33 (t, 3H, J=7.2 Hz), 1.24-1.30 (m, 4H), 0.97-1.06 (m, 1H).

Diethyl-(1′S,2′R,3′S,4′R,5′S)-4′-(6-aminopurin-9-yl)-2′,3′-O-(isopropylidene)-bicyclo[3.1.0]hexanephosphonate (23): Nucleoside 22 (100 mg, 0.22 mmol) was treated with 2 MNH₃ in i-PrOH (5 mL) and heated up to 70° C. After stirring for 19 h,the reaction mixture was evaporated to dryness. The resulting residuewas purified by silica gel column chromatography (0-6% MeOH in CH₂Cl₂,v/v) to afford nucleoside 23 (75 mg, 79%) as a white solid material.R_(f)=0.4 (5% MeOH in CH₂Cl₂, v/v); ESI-HRMS m/z 438.1912 [M+H]⁺,C₁₉H₂₈N₅O₅P.H⁺: Calcd. 438.1906); ¹H NMR (CDCl₃) δ8.38 (s, 1H), 8.36 (s,1H), 5.54 (s, 2H), 5.36 (d, 1H, J=7.2 Hz), 5.03 (s, 1H), 4.63 (d, 1H,J=7.2 Hz), 4.06-4.20 (m, 4H), 2.38 (t, 1H, J=16.5 Hz), 1.97-2.11 (m,1H), 1.78-1.85 (m, 1H), 1.68 (s, 3H), 1.32 (t, 3H, J=7.2 Hz), 1.27 (t,3H, J=7.2 Hz), 1.23 (s, 3H), 1.18-1.21 (m, 1H), 0.95-1.02 (m, 1H).

(1′S,2′R,3′S,4′R,5′S)-4′-(6-Aminopurin-9-yl)-2′,3′-(dihydroxy)-1′-(phosphonomethylene)-bicyclo[3.1.0]hexane(5): Nucleoside 23 (25 mg, 0.057 mmol) was coevaporated with anhydroustoluene (3×3 mL) and dissolved in anhydrous CH₂Cl₂ (3 mL).Iodotrimethylsilane (83 μl, 0.57 mmol) was added. After stirring for 15h, the reaction mixture was cooled to 0° C. followed by the addition ofice-cold H₂O (25 mL) and CH₂Cl₂ (25 mL). The phases were separated, andthe aqueous phase was washed with CH₂Cl₂ (1×35 mL) and diethyl ether(3×35 mL). The resulting aqueous phase was evaporated to dryness andpurified by HPLC (retention time: 17.5 min) to afford 5 (6.8 mg, 27%) asa white solid material. ESI-HRMS m/z 340.0817 [M−H]⁻, C₁₂H₁₅N₅O₅P⁻:Calcd. 340.0811); ¹H NMR (D₂O) δ 8.36 (s, 1H), 8.20 (s, 1H) 4.75 (s,1H), 4.63 (d, 1H, J=6.1 Hz), 4.09 (d, 1H, J=6.1 Hz), 3.19 (q, 6H, J=7.2Hz), 1.95-2.18 (m, 2H), 1.75-1.84 (m, 1H), 1.40-1.46 (m, 1H), 1.26 (t,9H, J=7.2 Hz), 0.92-1.02 (m, 1H). ³¹P NMR (D₂O) δ 25.36. Purity >99% byHPLC (retention time: 2.9 min).

(1′S,2′R,3′S,4′R,5′S)-4′-(2,6-Dichloropurin-9-yl)-1′-formyl-2,3-O-(isopropylidine)-bicyclo[3.1.0]hexane(25): Known nucleoside 24 (150 mg, 0.41 mmol) was coevaporated withanhydrous toluene (2×8 mL) and dissolved in anhydrous CH₂Cl₂ (8 mL).Dess-Martin periodinane (257 mg, 0.61 mmol) was added. After stirringfor 1 h, the reaction mixture was diluted with EtOAc (50 mL) and washedwith an aqueous mixture of Na₂S₂O₃ and NaHCO₃ (3×35 mL). The aqueousphase was then extracted with EtOAc (2×35 mL). The combined organicphase was evaporated to dryness, and the resulting residue was purifiedby silica gel column chromatography (0-100% EtOAc in petroleum ether,v/v) to afford compound 25 (120 mg, 80%) as a white solid material.R_(f)=0.6 (EtOAc); ESI-HRMS m/z 369.0527 ([M+H]⁺, C₁₅H₁₄Cl₂N₄O₃.H⁺:Calcd. 369.0521); ¹H NMR (CDCl₃) δ9.62 (s, 1H), 8.05 (s, 1H), 5.94 (d,1H, J=7.2 Hz), 4.97 (s, 1H), 4.83 (d, 1H, J=7.2 Hz), 2.22-2.29 (m, 1H),1.73 (t, 1H, J=6.1 Hz), 1.57 (s, 3H), 1.30 (s, 3H).

(1′S,2′R,3′S,4′R,5′S)-4′-(2,6-Dichloropurin-9-yl)-1′-[diisopropyl-(E)-ethenylphosphonate]-2′,3′-O-(isopropylidine)-bicyclo[3.1.0]hexane(26): Tetraisopropyl methylenediphosphonate (165 μL, 0.51 mmol) wasadded to a suspension of NaH (60% dispersion in mineral oil, 25 mg, 1.02mmol) in anhydrous THF (2 mL) at 0° C. After H₂ evolution ceased, asolution of aldehyde 25 (125 mg, 0.34 mmol) in anhydrous THF (3 mL) wasadded dropwise carefully at 0° C. After stirring at 0° C. for 1 h, themixture was warmed to rt. After stirring at rt for 1 h, the reactionmixture was cooled to 0° C., and ice-cold H₂O (20 mL) was added. Thephases were separated, and the aqueous phase was extracted with EtOAc(3×35 mL). The combined organic phase was evaporated to dryness, and theresulting residue was purified by silica gel column chromatography (0-4%MeOH in EtOAc, v/v) to afford nucleoside 26 (150 mg, 83%) as a whitesolid material. R_(f)=0.3 (EtOAc); ESI-HRMS m/z 531.1313 ([M+H]⁺,C₂₂H₂₉Cl₂N₄O₅P.H⁺: Calcd. 531.1331); ¹H NMR (CDCl₃) δ 8.04 (s, 1H),6.50-6.65 (m, 1H), 5.97 (t, 1H, J=17.1 Hz), 5.53 (d, 1H, J=7.2 Hz), 4.98(s, 1H), 4.77 (d, 1H, J=7.2 Hz), 4.60-4.74 (m, 2H), 1.82-1.90 (m, 1H),1.59 (s, 3H), 1.22-1.38 (m, 16H), 0.83-0.90 (m, 1H). ³¹P NMR (CDCl₃)δ16.64.

(1′S,2′R,3′S,4′R,5′S)-4′-(6-Amino-2-chloropurin-9-yl)-1′-[diisopropyl-(E)-ethenylphosphonate]-2,3-O-(isopropylidine)-bicyclo-[3.1.0]-hexane(27): Nucleoside 26 (100 mg, 0.19 mmol) was treated with 2 M NH₃ ini-PrOH (5 mL) and heated to 70° C. After stirring for 16 h, the reactionmixture was evaporated to dryness. The resulting residue was purified bysilica gel column chromatography (0-8% MeOH in CH₂Cl₂, v/v) to affordnucleoside 27 (85 mg, 88%) as a white solid material. R_(f)=0.3 (5% MeoHin EtOAc, v/v); ESI-HRMS m/z 512.1821 ([M+H]⁺, C₂₂H₃₁ClN₅O₅P.H⁺: Calcd.512.1830); ¹H NMR (CDCl₃) δ7.69 (s, 1H), 6.52-6.68 (m, 1H), 5.94 (t, 1H,J=17.5 Hz), 5.75 (s, 2H), 5.51 (d, 1H, J=7.2 Hz), 4.91 (s, 1H), 4.76 (d,1H, J=7.2 Hz), 4.59-4.73 (m, 2H), 1.80-1.89 (m, 1H), 1.61 (s, 3H),1.21-1.37 (m, 15H), 1.08-1.17 (m, 1H), 0.77-0.95 (m, 1H).

(1′S,2′R,3′S,4′R,5′S)-4′-(6-Amino-2-chloropurin-9-yl)-2′,3′-(dihydroxy)-1′-[(E)-phosphonoethenyl]-bicyclo[3.1.0]-hexane(7): Nucleoside 27 (12 mg, 0.023 mmol) was coevaporated with anhydroustoluene (3×2 mL) and dissolved in anhydrous CH₂Cl₂ (2 mL).Iodotrimethylsilane (35 μl, 0.24 mmol) was added. After stirring for 18h, the reaction mixture was cooled to 0° C., followed by the addition ofice-cold H₂O (15 mL) and CH₂Cl₂ (15 mL). The phases were separated, andthe aqueous phase was washed with CH₂Cl₂ (1×25 mL) and diethyl ether(3×35 mL). The resulting aqueous phase was evaporated to dryness andpurified by HPLC (retention time: 22.8 min) to afford 7 (2.5 mg, 28%) asa white solid material. ESI-HRMS m/z 386.0403 [M−H]⁻, C₁₃H₁₄N₅ClO₅P⁻:Calcd. 386.0421); ¹H NMR (D₂O) δ 7.99 (s, 1H), 6.21-6.36 (m, 1H), 6.06(t, 1H, J=17.5 Hz), 4.84-4.89 (m, 1H), 4.06 (d, 1H, J=6.6 Hz), 3.22 (q,3H, J=7.2 Hz), 1.99-2.06 (m, 1H), 1.78-1.87 (m, 1H), 1.29 (t, 6H, J=7.2Hz), 1.21-1.26 (m, 1H). ³¹P NMR (D₂O) δ 14.68. Purity >99% by HPLC(retention time: 4.3 min)

(1′S,2′R,3′S,4′R,5′S)-4′-(6-Aminopurin-9-yl)-1′-(diisopropyl-phosphonoethenyl)-2′,3′-O-(isopropylidine)-bicyclo[3.1.0]hexane(28): Nucleoside 27 (20 mg, 0.04 mmol) was dissolved in a mixture ofMeOH and aqueous 2 M NaOH (3 mL, 2:1, v/v). 10% Pd/C (20 mg) and H₂ (3bar) were added to this solution. After stirring the mixture for 19 h,the catalyst was removed by filtration through a Celite pad, which waswashed with MeOH (40 mL), and the filtrate was evaporated to dryness.The resulting residue was purified by silica gel column chromatography(0-10% MeOH in EtOAc, v/v) to afford nucleoside 28 (15 mg, 79%) as whitesolid material. R_(f)=0.5 (15% MeOH in EtOAc, v/v); ESI-HRMS m/z480.2385 ([M+H]⁺, C₂₂H₃₄N₅O₅P.H⁺: Calcd. 480.2376); ¹H NMR (CDCl₃) δ8.32 (s, 1H), 7.79 (s, 1H), 5.80 (s, 2H), 5.19 (d, 1H, J=7.2 Hz), 4.83(s, 1H), 4.74 (d, 1H, J=7.2 Hz), 4.63-4.73 (m, 2H), 1.60-2.35 (m, 4H),1.52 (s, 3H), 1.44-1.51 (m, 1H), 1.33 (s, 6H), 1.31 (s, 6H), 1.23 (s,3H), 1.04-1.09 (m, 1H), 0.76-0.83 (m, 1H). ³¹P NMR (CDCl₃) δ0.04.

(1′S,2′R,3′S,4′R,5′S)-4′-(6-Aminopurin-9-yl)-2′,3′-(dihydroxy)-1′-(phosphonoethenyl)-bicyclo[3.1.0]hexane(10): Nucleoside 28 (15 mg, 0.032 mmol) was coevaporated with anhydroustoluene (3×2 mL) and dissolved in anhydrous CH₂Cl₂ (2 mL).Iodotrimethylsilane (45 μl, 0.32 mmol) was added. After stirring for 15h, the reaction mixture was cooled to 0° C. followed by the addition ofice-cold H₂O (15 mL) and CH₂Cl₂ (15 mL). The phases were separated, andthe aqueous phase was washed with CH₂Cl₂ (1×25 mL) and diethyl ether(3×35 mL). The resulting aqueous phase was evaporated to dryness andpurified by HPLC (retention time: 16.6 min) to afford 10 (6.7 mg, 47%)as a white solid material. ESI-HRMS m/z 354.0970 [M−H]⁻, C₁₃H₁₇N₅O₅P⁻:Calcd. 354.0967); ¹H NMR (CDCl₃) δ 8.20 (s, 1H), 8.14 (s, 1H), 4.76 (s,1H), 4.58 (d, 1H, J=6.1 Hz), 4.09 (d, 1H, J=6.1 Hz), 3.21 (q, 3H, J=7.2Hz), 1.69-2.14 (m, 4H), 1.59-1.69 (m, 1H), 1.39-1.349 (m, 1H), 1.29 (t,6H, J=7.2 Hz), 0.81-0.92 (m, 1H). ³¹P NMR (D₂O) δ 27.95. Purity >99% byHPLC (retention time: 2.91 min)

(1S,2R,3S,4S,5S)-1-Formyl-2,3-O-(isopropylidene)-4-O-(tert-butyldimethylsilyl)-bicyclo[3.1.0]hexane(29): Compound 15 (0.63 g, 1.43 mmol) was coevaporated with anhydroustoluene (2×25 mL) and dissolved in anhydrous CH₂Cl₂ (25 mL). Dess-Martinperiodinane (0.91 g, 2.13 mmol) was added to this solution. Afterstirring for 4 h, the reaction mixture was diluted with EtOAc (50 mL)and washed with an aqueous mixture of Na₂S₂O₃ and NaHCO₃ (3×50 mL). Theaqueous phase was extracted with EtOAc (2×50 mL). The combined organicphase was evaporated to dryness and the resulting residue was purifiedby silica gel column chromatography (0-25% EtOAc in petroleum ether,v/v) to afford aldehyde 29 (452 mg, 73%) as a colorless oil. R_(f)=0.6(50% EtOAc in petroleum ether, v/v); ESI-HRMS m/z 459.1986 ([M+Na]⁺,C₂₆H₃₂O₄Si.Na⁺: Calcd. 459.1968); ¹H NMR (CDCl₃) δ8.92 (s, 1H),7.68-7.78 (m, 4H, Ph), 7.31-7.48 (m, 6H, Ph), 5.13 (d, 1H, J=6.5 Hz),4.41 (t, 1H, J=6.5 Hz), 4.16 (t, 1H, J=6.5 Hz), 2.19-2.28 (m, 1H),2.10-2.18 (m, 1H), 1.55 (s, 3H), 1.43-1.51 (m, 1H), 1.23 (s, 3H), 1.09(s, 9H).

(1S,2R,3S,4S,5S)-1-[Diisopropyl-(E)-phosphonoethenyl]-2,3-O-(isopropylidene)-4-O-(tert-butyldimethylsilyl)-bicyclo[3.1.0]hexane(30): Tetraisopropyl methylenediphosphonate (475 μL, 1.47 mmol) wasadded to a suspension of sodium hydride (71 mg, 2.95 mmol, 60%dispersion in mineral oil) in anhydrous THF (6 mL) at 0° C. After H₂evolution ceased, a solution of aldehyde 29 (0.43 g, 0.98 mmol) inanhydrous THF (4 mL) was added dropwise carefully at 0° C. Afterstirring at 0° C. for 1 h, the mixture was warmed to rt. After stirringat rt for 1 h, the mixture was cooled to 0° C., and ice-cold H₂O (20 mL)was added. The phases were separated, and the aqueous phase wasextracted with EtOAc (3×35 mL). The combined organic phase wasevaporated to dryness and the resulting residue was purified by silicagel column chromatography (0-70% EtOAc in petroleum ether, v/v) toafford nucleoside 30 (0.24 mg, 48%) as a white solid material. R_(f)=0.4(70% EtOAc in petroleum ether, v/v); ESI-HRMS m/z 599.2938 ([M+H]⁺,C₃₃H₄₇O₆PSi.H⁺: Calcd. 599.2958); ¹H NMR (CDCl₃) δ 7.69-7.77 (m, 4H,Ph), 7.31-7.45 (m, 6H, Ph), 6.24-6.40 (m, 1H), 5.66 (t, 1H, J=17.5 Hz),4.75 (d, 1H, J=6.5 Hz), 4.52-4.64 (m, 2H) 4.42 (t, 1H, J=6.5 Hz), 4.11(t, 1H, J=6.5 Hz), 1.93-1.99 (m, 1H), 1.78-1.85 (m, 1H), 1.57 (s, 3H),1.19-1.32 (m, 15H), 1.07 (s, 9H), 0.93-1.01 (m, 1H).

(1S,2R,3S,4S,5S)-1-[Diisopropyl-(E)-phosphonoethenyl]-4-(hydroxy)-2,3-O-(isopropylidene)-bicyclo[3.1.0]hexane(31): Compound 30 (0.45 g, 0.76 mmol) was dissolved in THF (10 mL) andtetrabutylammonium fluoride (1.0 M in THF, 2.3 mL, 2.3 mmol) was added.After stirring for 13 h, the reaction mixture was evaporated to dryness.The resulting residue was purified by silica gel column chromatography(0-7% MeOH in EtOAc, v/v) to afford compound 31 (0.26 g, 98%) as acolorless oil. R_(f)=0.3 (EtOAc); ESI-HRMS m/z 361.1790 ([M+H]⁺,C₁₇H₂₉O₆P.H⁺: Calcd. 361.1780); ¹H NMR (CDCl₃) δ6.34-6.49 (m, 1H), 5.74(t, 1H, J=17.5 Hz), 4.99 (d, 1H, J=6.5 Hz), 4.47-4.69 (m, 4H), 2.40 (d,1H, J=9.5 Hz), 2.07-2.15 (M, 1H), 1.59-1.63 (m, 1H), 1.58 (s, 3H),1.22-1.34 (m, 15H), 0.93-1.07 (m, 1H).

(1′S,2′R,3′S,4′R,5′S)-4′-(6-Chloropurin-9-yl)-1′-[diisopropyl-(E)-phosphonoethenyl]-2′,3′-O-(isopropylidene)-bicyclo[3.1.0]hexane(32): Diisopropyl azodicarboxylate (90 μL, 0.45 mmol) was added at rt toa mixture of triphenylphosphine (117 mg, 0.45 mmol) and 6-chloropurine(70 mg, 0.45 mmol) in anhydrous THF (5 mL). After stirring for 30 min, asolution of the compound 31 (80 mg, 0.23 mmol) in THF (5 mL) was added.After stirring for 60 h, the reaction mixture was evaporated to dryness.The resulting residue was purified by silica gel column chromatography(0-55% acetone in petroleum ether, v/v) to afford nucleoside 32 (92 mg,85%) as a white solid material. R_(f)=0.4 (60% acetone in petroleumether, v/v); ESI-HRMS m/z 519.1532 ([M+Na]⁺, C₂₂H₃₀ClN₄O₅P.Na⁺: Calcd.519.1540); ¹H NMR (CDCl₃) δ 8.71 (s, 1H), 8.07 (s, 1H), 6.49-6.63 (m,1H), 5.96 (t, 1H, J=17.5), 5.53 (d, 1H, J=6.5 Hz), 5.02 (s, 1H), 4.79(d, 1H, J=6.5 Hz), 4.61-4.73 (m, 2H), 1.86-1.92 (m, 1H), 1.58-1.63 (m,1H), 1.54 (s, 3H), 1.22-1.38 (m, 16H).

(1′S,2′R,3′S,4′R,5′S)-4′-(6-Aminopurin-9-yl)-1′-[diisopropyl-(E)-phosphonoethenyl]-2′,3′-O-(isopropylidene)-bicyclo[3.1.0]hexane(33): Nucleoside 32 (90 mg, 0.19 mmol) was treated with 2 M NH₃ ini-PrOH (7 mL) and heated to 70° C. After stirring for 17 h, the reactionmixture was evaporated to dryness. The resulting residue was purified bysilica gel column chromatography (0-12% MeOH in CH₂Cl₂, v/v) to affordnucleoside 33 (74 mg, 85%) as a white solid material. R_(f)=0.2 (10%MeOH in EtOAc, v/v); ESI-HRMS m/z 478.2198 ([M+H]⁺, C₂₂H₃₂N₅O₅P.H⁺:Calcd. 478.2219); ¹H NMR (CDCl₃) δ 8.30 (s, 1H), 7.73 (s, 1H), 6.51-6.66(m, 1H), 5.96 (t, 1H, J=17.5), 5.47-5.54 (m, 3H), 4.95 (s, 1H), 4.78 (d,1H, J=6.5 Hz), 4.60-4.72 (m, 2H), 1.86-1.94 (m, 1H), 1.53-1.57 (m, 4H),1.24-1.37 (m, 16H).

(1′S,2′R,3′S,4′R,5′S)-4′-(6-Aminopurin-9-yl)-2′,3′-(dihydroxy)-1′-[(E)-phosphonoethenyl]-bicyclo[3.1.0]hexane(8): Nucleoside 33 (20 mg, 0.042 mmol) was coevaporated with anhydroustoluene (3×5 mL) and dissolved in anhydrous CH₂Cl₂ (5 mL).Iodotrimethylsilane (60 μl, 0.42 mmol) was added. After stirring for 17h, the reaction mixture was cooled to 0° C., followed by the addition ofice-cold H₂O (25 mL) and CH₂Cl₂ (25 mL). The phases were separated andthe aqueous phase was washed with CH₂Cl₂ (1×35 mL) and diethyl ether(3×35 mL). The resulting aqueous phase was evaporated to dryness andpurified by HPLC (retention time: 16.5 min) to afford 8 (11.8 mg, 78%)as a white solid material. ESI-HRMS m/z 352.0821 [M−H]⁻, C₁₃H₁₅N₅O₅P⁺:Calcd. 352.0811); ¹H NMR (D₂O) δ 8.30 (s, 1H), 8.06 (s, 1H), 6.30-6.44(m, 1H), 6.07 (t, 1H, J=17.5), 4.97 (s, 1H), 4.89 (d, 1H, J=7.2 Hz),4.09 (d, 1H, J=7.2 Hz), 3.21 (q, 3H, J=7.2 Hz), 2.03-2.10 (m, 2H),1.84-1.89 (m, 1H), 1.29 (t, 7H, J=7.2 Hz). ³¹P NMR (D₂O) δ 15.71.Purity >99% by HPLC (retention time: 3.5 min)

(1S,2R,3S,4S,5S)-1-(Diisopropyl-phosphonoethenyl)-4-(hydroxy)-2,3-O-(isopropylidene)-bicyclo[3.1.0]hexane(34): Compound 31 (30 mg, 0.083 mmol) was dissolved in MeOH (3 mL). 10%Pd/C (25 mg) and H₂ (3 bar) was added. After stirring the mixture for 17h, the catalyst was removed by filtration through a Celite pad, whichwas washed with MeOH (40 mL), and the filtrate was evaporated todryness. The resulting residue was purified by silica gel columnchromatography (0-90% acetone in petroleum ether, v/v) to affordnucleoside 34 (22 mg, 72%) as white solid material. R_(f)=0.3 (5% MeOHin EtOAc, v/v); ESI-HRMS m/z 363.1933 ([M+H]⁺, C₁₇H₃₁O₆P.H⁺: Calcd.363.1937); ¹H NMR (CDCl₃) δ 4.61-4.76 (m, 2H), 4.43-4.54 (m, 2H),4.17-4.35 (m, 1H), 2.32 (d, J=9.8 Hz, 1H), 1.43-1.93 (m, 8H), 1.22-1.37(m, 15H), 1.07-1.14 (m, 1H), 0.47-0.56 (m, 1H).

(1′S,2′R,3′S,4′R,5′S)-4′-(2,6-Dichloropurin-9-yl)-1′-(diisopropyl-phosphonoethenyl)-2′,3′-O-(isopropylidene)-bicyclo[3.1.0]hexane(35): Diisopropyl azodicarboxylate (93 μL, 0.47 mmol) was added at rt toa mixture of triphenylphosphine (123 mg, 0.47 mmol) and2,6-dichloropurine (89 mg, 0.47 mmol) in anhydrous THF (4 mL). Afterstirring for 30 min, a solution of the compound 34 (85 mg, 0.24 mmol) inTHF (4 mL) was added. After stirring for 65 h, the reaction mixture wasevaporated to dryness. The resulting residue was purified by silica gelcolumn chromatography (0-5% MeOH in EtOAc, v/v) to afford nucleoside 35(50 mg, 40%) as a white solid material. R_(f)=0.4 (5% MeOH in EtOAc,v/v); ESI-HRMS m/z 533.1497 ([M+H]⁺, C₂₂H₃₁Cl₂N₄O₅P.H⁺: Calcd.533.1487); ¹H NMR (CDCl₃) δ8.09 (s, 1H), 5.20 (d, 1H, J=7.2 Hz), 4.86(s, 1H), 4.73 (d, 1H, J=7.2 Hz), 4.63-4.73 (m, 2H), 2.25-2.44 (m, 1H),1.79-2.09 (m, 2H), 1.58-1.70 (m, 1H), 1.52 (s, 3H), 1.43-1.52 (m, 1H),1.28-1.35 (m, 12H), 1.24 (s, 3H), 1.04-1.11 (m, 1H), 0.78-0.87 (m, 1H).

(1′S,2′R,3′S,4′R,5′S)-4′-(6-Amino-2-chloropurin-9-yl)-1′-(diisopropyl-phosphonoethenyl)-2′,3′-O-(isopropylidene)-bicyclo[3.1.0]hexane(36): Nucleoside 35 (50 mg, 0.094 mmol) was treated with 2 M NH₃ ini-PrOH (5 mL) and heated to 70° C. After stirring for 19 h, the reactionmixture was evaporated to dryness. The resulting residue was purified bysilica gel column chromatography (0-10% MeOH in CH₂Cl₂, v/v) to affordnucleoside 36 (34 mg, 71%) as a white solid material. R_(f)=0.4 (8% MeOHin EtOAc, v/v); ESI-HRMS m/z 514.1978 ([M+H]⁺, C₂₂H₃₃ClN₅O₅P.H⁺: Calcd.514.1986); ¹H NMR (CDCl₃) δ7.73 (s, 1H), 5.84 (s, 2H), 5.20 (d, 1H,J=6.5 Hz), 4.76 (s, 1H), 4.72 (d, 1H, J=6.5 Hz), 4.62-4.71 (m, 2H),2.24-2.40 (m, 1H), 1.75-2.08 (m, 1H), 1.56-1.74 (m, 5H), 1.40-1.47 (m,1H), 1.28-1.35 (m, 12H), 1.24 (s, 3H), 1.01-1.06 (m, 1H), 0.74-0.82 (m,1H).

(1′S,2′R,3′S,4′R,5′S)-4-(6-Amino-2-chloropurin-9-yl)-2′,3′-(dihydroxy)-1′-(phosphonoethenyl)-bicyclo[3.1.0]hexane(9): Nucleoside 23 (25 mg, 0.049 mmol) was coevaporated with anhydroustoluene (3×4 mL) and dissolved in anhydrous CH₂Cl₂ (4 mL).Iodotrimethylsilane (70 μl, 0.49 mmol) was added. After stirring for 19h, the reaction mixture was cooled to 0° C. followed by the addition ofice-cold H₂O (25 mL) and CH₂Cl₂ (25 mL). The phases were separated, andthe aqueous phase was washed with CH₂Cl₂ (1×35 mL) and diethyl ether(3×35 mL). The resulting aqueous phase was evaporated to dryness andpurified by HPLC (retention time: 21.5 min) to afford 9 (8.5 mg) and 10(1.3 mg, 53%, combined yield) as a white solid materials. ESI-HRMS m/z388.0574 [M−H]⁻, C₁₃H₁₆ClN₅O₅P⁻: Calcd. 388.0578); ¹H NMR (D₂O) δ 8.15(s, 1H), 4.74 (s, 1H), 4.61 (d, 1H, J=7.2 Hz), 4.11 (d, 1H, J=7.2 Hz),3.21 (q, 3H, J=7.2 Hz), 1.70-2.11 (m, 4H), 1.63-1.71 (m, 1H), 1.33-1.38(m, 1H), 1.29 (t, 3H, J=7.2 Hz), 0.81-0.91 (m, 1H). ³¹P NMR (D₂O) δ28.26. Purity >99% by HPLC (retention time: 4.6 min)

(1S,2R,3S,4S,5S)-1-Bromomethyl-2,3-O-(isopropylidene)-4-O-(tert-butyldimethylsilyl)-bicyclo[3.1.0]hexane(37): Compound 15 (0.30 g, 0.69 mmol) was coevaporated with anhydroustoluene (3×10 mL) and dissolved in anhydrous CH₂Cl₂ (8 mL). CBr₄ (0.46g, 1.36 mmol) triphenylphosphine (0.36 g, 1.36 mmol), and triethylamine(0.3 mL, 2.07 mmol) were added. After stirring for 17 h, the reactionmixture was diluted with CH₂Cl₂ (50 mL) and sat. aqueous NaCl (25 mL).The phases were separated and aqueous phase was extracted with CH₂Cl₂(3×25 mL). The combined organic phase was evaporated to dryness and theresulting residue was purified by silica gel column chromatography(0-20% EtOAc in petroleum ether, v/v) to afford compound 37 (0.28 g,81%) as a colorless oil. R_(f)=0.8 (50% EtOAc in petroleum ether, v/v);ESI-HRMS m/z 523.1296 ([M+Na]⁺, C₂₆H₃₃BrO₃Si.Na⁺: Calcd. 523.1280); ¹HNMR (CDCl₃) δ 7.67.77 (m, 4H, Ph), 7.30-7.45 (m, 6H, Ph), 4.77 (d, 1H,J=6.5 Hz), 4.44 (t, 1H, J=6.5 Hz), 4.08 (t, 1H, J=6.5 Hz), 3.76 (d, 1H,J=10.5 Hz), 3.13 (d, 1H, J=10.5 Hz), 1.80-1.87 (m, 1H), 1.60-1.70 (m,1H), 1.55 (s, 3H), 1.21 (s, 3H), 1.05 (s, 9H), 0.71-0.86 (m, 1H).

(1S,2R,3S,4S,5S)-1-C-(Ethoxymethylphosphinyl)-2,3-O-(isopropylidene)-4-O-(tert-butyldimethylsilyl)-bicyclo[3.1.0]hexane(38): Compound 37 (0.28 g, 0.56 mmol) was dissolved indiethylmethylphosphite (4 mL) and heated up to 110° C. After stirringfor 17 h, the reaction mixture was cooled to rt and evaporated todryness. The resulting residue was purified by silica gel columnchromatography (0-90% EtOAc in petroleum ether, v/v) to affordinseparable diastereomeric mixture of compound 38 (0.28 g, 95%) as acolorless oil. R_(f)=0.6 (5% MeOH in EtOAc, v/v); ESI-HRMS m/z 529.2532([M+H]⁺, C₂₉H₄₁O₅PSi.H⁺: Calcd. 529.2539); ¹H NMR (CDCl₃) δ 7.68-7.77(m, 6.8H, Ph), 7.29-7.46 (m, 10.2H, Ph), 4.75 (d, 0.7H, J=6.5 Hz), 4.68(d, 1H, J=6.5 Hz), 4.43-4.49 (m, 1.7H), 3.87-4.14 (m, 5.1H), 1.73-1.92(m, 3.4H), 1.62 (s, 5.1H), 1.57-1.60 (m, 1.7H), 1.48 (d, 3H, J=3.4 Hz),1.44 (d, 2.1H, J=3.4 Hz), 1.26 (t, 3H, J=7.2 Hz), 1.22 (t, 2.1H, J=7.2Hz), 1.19 (s, 2.1H), 1.18 (s, 3H), 1.09-1.13 (m, 1.7H), 1.07 (s, 15.3H),0.52-0.69 (m, 1H).

(1S,2R,3S,4S,5S)-1-C-(Ethoxymethylphosphinyl)-4-hydroxy-2,3-O-(isopropylidene)-bicyclo[3.1.0]hexane(39): Compound 38 (0.30 g, 0.57 mmol) was dissolved in THF (10 mL) andtetrabutylammonium fluoride (1.0 M in THF, 1.70 mL, 1.70 mmol) wasadded. After stirring for 21 h, the reaction mixture was evaporated todryness. The resulting residue was purified by silica gel columnchromatography (0-15% MeOH in EtOAc, v/v) to afford an inseparablediastereomeric mixture of compound 39 (0.15 g, 91%) as a colorless oil.R_(f)=0.2 (15% MeOH in CH₂Cl₂, v/v); ESI-HRMS m/z 291.1366 ([M+H]⁺,C₁₃H₂₃O₅P.H⁺: Calcd. 291.1361); ¹H NMR (CDCl₃) δ 4.89-5.03 (m, 2H),4.50-4.60 (m, 4H), 3.97-4.14 (m, 4H), 2.34-2.40 (m, 2H), 1.95 (t, 2H,J=7.7 Hz), 1.90 (t, 2H, J=7.7 Hz), 1.78-1.87 (m, 2H), 1.66 (s, 6H), 1.55(d, 3H, J=3.9 Hz), 1.50 (d, 3H, J=3.9 Hz), 1.29-1.35 (m, 6H), 1.28 (s,6H), 1.23-1.26 (m, 2H), 0.60-0.71 (m, 2H).

(1′S,2′R,3′S,4′R,5′S)-1′-C-(Ethoxymethylphosphinyl)-4′-(2,6-dichloropurin-9-yl)-2′,3′-O-(isopropylidene)-bicyclo[3.1.0]hexane(40): Diisopropyl azodicarboxylate (360 μL, 1.82 mmol) was added at rtto a mixture of triphenylphosphine (0.48 g, 1.82 mmol) and2,6-dichloropurine (0.35 g, 1.82 mmol) in anhydrous THF (5 mL). Afterstirring for 30 min, a solution of the compound 39 (0.27 g, 0.91 mmol)in THF (5 mL) was added. After stirring for 60 h, the reaction mixturewas evaporated to dryness. The resulting residue was purified by silicagel column chromatography (0-10% MeOH in EtOAc, v/v) to affordinseparable diastereomeric mixture of nucleoside 40 (0.25 mg, 60%) as awhite solid material. R_(f)=0.2 (10% MeOH in EtOAc, v/v); ESI-HRMS m/z461.0899 ([M+H]⁺, C₁₈H₂₃Cl₂N₄O₄P.H⁺: Calcd. 461.0912); ¹H NMR (CDCl₃) δ8.79 (s, 0.5H), 8.51 (s, 1H), 5.45 (d, 0.5H, J=7.2 Hz), 5.32 (d, 1H,J=7.2 Hz), 5.06 (s, 0.5H), 4.96 (s, 1H), 4.66 (d, 1.5H, J=7.2 Hz),3.98-4.21 (m, 3H), 2.40-2.51 (m, 0.5H), 2.15-2.24 (m, 1H), 1.93-2.11 (m,1.5H), 1.63-1.75 (m, 0.5H), 1.61 (s, 5.5H), 1.59 (d, 3H, J=3.9 Hz), 1.55(d, 1.5H, J=3.9 Hz), 1.35 (t, 3H, J=7.2 Hz), 1.25 (t, 1.5H, J=7.2 Hz),1.24 (s, 4.5H), 1.18-1.23 (m, 1.5H), 0.95-1.13 (m, 1.5H).

(1′S,2′R,3′S,4′R,5′S)-4′-(6-Amino-2-chloropurin-9-yl)-1′-C-(ethoxymethylphosphinyl)-2′,3′-O-(isopropylidene)-bicyclo[3.1.0]hexane(41): Nucleoside 40 (0.20 g, 0.44 mmol) was treated with 2M NH₃ ini-PrOH (8 mL) and heated to 70° C. After stirring for 15 h, the reactionmixture was evaporated to dryness. The resulting residue was purified bysilica gel column chromatography (0-7% MeOH in CH₂Cl₂, v/v) to affordnucleoside 41 (150 mg, 79%) as a white solid material. R_(f)=0.4 (10%MeOH in CH₂Cl₂, v/v); ESI-HRMS m/z 442.1416 ([M+H]⁺, C₁₈H₂₆ClN₅O₄P.H⁺:Calcd. 442.1411); ¹H NMR (CDCl₃) δ 8.21 (s, 0.5H), 8.00 (s, 1H), 5.96(s, 3H), 5.40 (d, 0.5H, J=6.5 Hz), 5.30 (d, 1H, J=6.5 Hz), 4.91 (s,0.5H), 4.81 (s, 1H), 4.62-4.71 (d, 1.5H, J=7.2 Hz), 4.10-4.22 (m, 2H),3.98-4.09 (m, 1H), 3.60-3.80 (m, 1H), 2.64 (t, 1H, J=15.3 Hz), 2.35 (t,0.5H, J=15.3 Hz), 2.01-2.17 (m, 0.5H), 1.87 (t, 1.5H, J=15.8 Hz), 1.75(s, 4.5H), 1.55-1.69 (m, 4.5H), 1.36 (t, 3H, J=7.2 Hz), 1.25 (t, 1.5H,J=7.2 Hz), 1.24 (s, 4.5H), 1.17-1.22 (m, 1.5H), 0.96-1.07 (m, 1.5H).

(1′S,2′R,3′S,4′R,5′S)-4′-(6-Amino-2-chloropurin-9-yl)-2′,3′-dihydroxy-1′-(methylphosphonicacid)-bicyclo[3.1.0]hexane(11) and (1′S,2′R,3′S,4′R,5′S)-4′-(6-Aminopurin-9-yl)-2′,3′-dihydroxy-1′-(methylphosphonicacid)-bicyclo[3.1.0]hexane(12): Nucleoside 29 (15 mg, 0.034 mmol) was coevaporated with anhydroustoluene (3×3 mL) and dissolved in anhydrous CH₂Cl₂ (4 mL).Iodotrimethylsilane (91 μl, 0.33 mmol) was added. After stirring for 19h, the reaction mixture was cooled to 0° C., followed by the addition ofice-cold H₂O (25 mL) and CH₂Cl₂ (25 mL). The phases were separated andthe aqueous phase was washed with CH₂Cl₂ (1×35 mL) and diethyl ether(3×35 mL). The resulting aqueous phase was evaporated to dryness andpurified by HPLC (retention time: 16.8 min) to afford 11 (1.3 mg, 11%)and 12 (0.8 mg, 20%, combined yield) as a white solid material.

Analytical data of compound 11: ESI-HRMS m/z 372.0625 [M−H]⁻,C₁₃H₁₆ClN₅O₄P⁻: Calcd. 372.0628); ¹H NMR (D₂O) δ 8.23 (s, 1H), 4.76-4.79(m, 1H), 4.63 (d, 1H, J=6.2 Hz), 4.12 (d, 1H, J=6.2 Hz), 3.21 (q, 2H,J=7.2 Hz), 2.34 (t, 1H, J=15.3 Hz), 1.75-1.86 (m, 1H), 1.68-1.75 (m,1H), 1.51-1.56 (m, 1H) 1.35 (d, 3H, J=13.2 Hz), 1.26 (t, 1H, J=7.2 Hz)0.96-1.04 (m, 1H). ³¹P NMR (D₂O) δ 46.01. Purity >99% by HPLC (retentiontime: 4.19 min).

Analytical data of compound 12: ESI-HRMS m/z 338.1016 [M−H]⁻,C₁₃H₁₇N₅O₄P⁻: Calcd. 338.1018); ¹H NMR (D₂O) δ 8.26 (s, 1H), 8.25 (s,1H), 4.76-4.79 (m, 1H), 4.63 (d, 1H, J=6.2 Hz), 4.12 (d, 1H, J=6.2 Hz),3.21 (q, 2H, J=7.2 Hz), 2.34 (t, 1H, J=15.3 Hz), 1.75-1.86 (m, 1H),1.68-1.75 (m, 1H), 1.51-1.56 (m, 1H) 1.35 (d, 3H, J=13.2 Hz), 1.26 (t,1H, J=7.2 Hz) 0.96-1.04 (m, 1H). ³¹P NMR (D₂O) δ 46.0. Purity >99% byHPLC (retention time: 5.91 min).

Experimental Procedures for Example 3

General methods: Compound 13a was either synthesized as reported orobtained as a custom synthesis from Natland International Corporation(Research Triangle Park, N.C.). All other reagents and solvents (regularand anhydrous) were of analytical grade and obtained from commercialsuppliers and used without further purification. Reactions wereconducted under an atmosphere of argon whenever anhydrous solvents wereused. All reactions were monitored by thin-layer chromatography (TLC)using silica gel coated plates with a fluorescence indicator which werevisualized: a) under UV light, b) by dipping in 5% conc. H₂SO₄ inabsolute ethanol (v/v) followed by heating, or c) by dipping in asolution of anisaldehyde:H₂SO₄ (1:2, v/v) in MeOH followed by heating.Silica gel column chromatography was performed with silica gel (SiO₂,200-400 mesh, 60A) using moderate air pressure. Evaporation of solventswas carried out under reduced pressure at a temperature below 50° C.After column chromatography, appropriate fractions were pooled,evaporated and dried at high vacuum for at least 12 h to give theobtained products in high purity. ¹H NMR and ³¹P NMR ascertained samplepurity. No corrections in yields were made for solvent ofcrystallization. ¹H NMR and ³¹P NMR spectra were recorded at 300 MHz and121.5 MHz, respectively. Chemical shifts are reported in parts permillion (ppm) relative to tetramethylsilane or deuterated solvent as theinternal standard (²H: CDCl₃ 7.26 ppm). Systematic compound names forbicyclic nucleosides are given according to the von Baeyer nomenclature.High resolution mass spectroscopic (HRMS) measurements were performed ona proteomics optimized Q-TOF-2 (Micromass-Waters) using externalcalibration with polyalanine. Observed mass accuracies are thoseexpected on the basis of known performance of the instrument as well asthe trends in masses of standard compounds observed at intervals duringthe series of measurements. Reported masses are observed massesuncorrected for this time-dependent drift in mass accuracy.

Purification of the nucleotide derivatives for biological testing wasperformed by HPLC with a Luna 5 micron RP-C18 semipreparative column(250×10.0 mm; Phenomenex, Torrance, Calif.) under the followingconditions: flow rate of 2 mL/min; 10 mM triethylammonium acetate(TEAA)-CH₃CN from 100:0 (v/v) to 70:30 (v/v) in 30 min and isolated inthe triethylammonium salt form. Analytical purity of compounds waschecked using a Hewlett-Packard 1100 HPLC equipped with Zorbax SB-Aq 5μm analytical column (50×4.6 mm; Agilent Technologies Inc, Palo Alto,Calif.). Mobile phase: linear gradient solvent system: 5 mM TBAP(tetrabutylammonium dihydrogenphosphate)-CH₃CN from 80:20 to 40:60 in 13min; the flow rate was 0.5 mL/min. Peaks were detected by UV absorptionwith a diode array detector at 254, 275, and 280 nm. All derivativestested for biological activity showed >99% purity by HPLC analysis(detection at 254 nm).

(1′S,2′R,3′S,4′R,5′S)-4-(6-amino-2-chloro-purin-9-yl)-2,3-(dihydroxyl)-1-[hydroxymethyl]bicyclo-[3.1.0]hexane(19a). Nucleoside 18a (25 mg, 0.071 mmol) was dissolved in 10% aqueoustrifluoroacetic acid (1.5 mL, v/v). After stirring at room temperaturefor 17 h, the reaction mixture was evaporated to dryness. The resultingresidue was purified by silica gel column chromatography (0-12% MeOH inCH₂Cl₂, v/v) to afford 2′,3′,5′-trihydroxy nucleoside 19a (15.2 mg,69%). R_(f)=0.3 (20% MeoH in CH₂Cl₂, v/v). ¹H NMR (MeOD-d₄) δ 8.48 (s,1H), 4.80 (s, 1H), 4.76 (d, J=7.1 Hz, 1H), 4.23-4.28 (d, J=11.5 Hz, 1H),3.87 (d, J=7.1 Hz, 1H), 3.35 (s, 1H), 1.58-1.63 (m, 1H), 1.50-1.55 (m,1H), 0.72-0.78 (m, 1H).

(1′S,2′R,3′S,4′R,5′S)-4-(6-amino-2-chloro-purin-9-yl)-1-[iodomethyl]-2,3-(O-isopropylidine)bicyclo-[3.1.0]hexane (20a). Nucleoside 18a (45 mg, 0.128 mmol) wascoevaporated with anhydrous toluene (3×10 mL) and dissolved in anhydrousTHF (3 mL). I₂ (66 mg, 0.256 mmol), triphenylphosphine (68 mg, 0.256mmol), and imidazole (18 mg, 0.256 mmol) were added. After stirring for17 h, the reaction mixture was diluted with EtOAc (30 mL) and washedwith saturated aqueous Na₂S₂O₃ (2×15 mL). The phases were separated andaqueous phase was extracted with EtOAc (3×25 mL). The combined organicphase was evaporated to dryness, and the resulting residue was purifiedby silica gel column chromatography (0-80% EtOAc in petroleum ether,v/v) to afford 5′-iodo nucleoside 20a (41 mg, 74%). R_(f)=0.4 (5% MeoHin CH₂Cl₂, v/v). ESI-HRMS m/z 462.0205 ([M+H]⁺ (C₁₅H₁₈N₅O₂Cl, calcd462.0194). ¹H NMR (CDCl₃) δ 8.09 (s, 1H), 5.91 (s, 2H), 5.32 (d, 1H,J=7.2 Hz), 4.91 (s, 1H), 5.32 (d, 1H, J=7.2 Hz), 3.63-3.69 (d, 1H,J=10.5 Hz), 3.53-3.56 (d, 1H, J=10.5 Hz), 1.65-1.74 (m, 1H), 1.71 (s,3H), 1.29 (s, 3H), 1.22-1.31 (m, 1H), 1.10-1.15 (m, 1H).

(1′S,2′R,3′S,4′R,5′S)-4-(6-amino-2-chloro-purin-9-yl)-2,3-(dihydroxy)-1-[iodomethyl]bicyclo-[3.1.0]hexane(21a). 5-Iodo nucleoside 20a (106 mg, 0.229 mmol) was dissolved in THF(1 mL), followed by the addition of 10% aqueous trifluoroacetic acid(3.5 mL, v/v). After stirring the reaction mixture at 65° C. for 15 h,the reaction mixture was evaporated to dryness and the resulting residuewas purified by silica gel column chromatography (0-80% EtOAc inpetroleum ether, v/v) to afford 2′,3′-dihydroxy-5′-iodo nucleoside 21a(41 mg, 60%). R_(f)=0.3 (10% MeOH in CH₂Cl₂, v/v). ESI-HRMS m/z 421.9883([M+H]⁺ (C₁₂H₁₄N₅O₂ClI, calcd 421.9881). ¹H NMR (MeOD-d₄) δ 8.41 (s,1H), 4.75 (dd, 1H, J=1.8 Hz), 4.10 (dt, 1H, J=8 Hz, 2.8 Hz), 3.87-3.91(d, 1H, J=10.5 Hz), 3.46-3.52 (d, 1H, J=10.5 Hz), 1.91-1.95 (m, 1H),1.67-1.72 (m, 1H), 1.04-1.09 (m, 1H).

(1′S,2′R,3′S,4′R,5′S)-4-(6-amino-2-chloro-purin-9-yl)-2,3-(dihydroxy)-1-[monophosphorothioate]-bicyclo-[3.1.0]hexane(12a). To the suspension of 5′-iodo nucleoside 21a (3 mg, 7.12 μmol) andH₂O (0.5 mL), trisodium thiophosphate (10 mg, 55 μmol) was added. Afterstirring the reaction mixture for 3 d at room temperature under argonatmosphere, the reaction mixture was lyophilized and purified by semipreparative HPLC (retention time 19.5 min) to get5′-monophosphorothioate 12a (1.65 mg, 57%) as a white solid. ESI-HRMSm/z 406.0159 ([M+H]⁺ (C₁₉H₉N₅O₂SCl, calcd 406.0165). ¹H NMR (D₂O) δ 8.39(s, 1H), 4.62 (s, 1H), 4.63 (s, 1H), 3.97 (d, 1H, J=6.5 Hz), 3.19-3.26(m, 1H), 2.79-2.87 (m, 1H), 1.69-1.74 (m, 1H), 1.39-1.43 (m, 1H),0.84-0.90 (m, 1H).

Ethyl(1′S,2′R′3′S,4′R,5′S)-4-(6-amino-2-chloropurin-9-yl]-2′,3′-O-(isopropylidene)-bicyclo[3.1.0]hexanecarboxylate(23a). Nucleoside 22a (53 mg, 0.13 mmol) was treated with 2 M NH₃ ini-PrOH (5 mL) and heated to 70° C. After stirring the reaction for 16 h,the reaction mixture was evaporated to dryness. The resulting residuepurified by silica gel column chromatography (0-4% MeOH in CH₂Cl₂, v/v)to afford nucleoside 23a (35 mg, 68%) as a white solid. R_(f)=0.4 (5%MeoH in CH₂Cl₂, v/v). ESI-HRMS m/z 394.1286 ([M+H]⁺ (C₁₇H₂₁N₅O₄Cl, calcd394.1282). ¹H NMR (MeOD-d₄) δ 8.09 (s, 1H), 5.85 (d, 1H, J=7.1 Hz), 4.98(s, 1H), 4.81 (d, 1H, J=7.1 Hz), 4.20-4.26 (m, 1H), 2.23-2.28 (m, 1H),1.64-1.67 (m, 1H), 1.52 (s, 3H), 1.34 (t, 3H, J=7.2 Hz), 1.20-1.29 (m,4H).

(1′S,2′R,3′S,4′R,5′S)-4-(6-amino-2-chloro-purin-9-yl)-1-[hydroxydeuteromethyl]-2,3-(O-isopropylidine)bicyclo-[3.1.0]hexane(24a). Nucleoside 23a (9 mg, 23 μmol) was coevaporated with anhydroustoluene (3×10 mL), and dissolved in anhydrous THF (10 mL). LiBD₄ (3 mg,115 μmol) was added and after stirring the reaction mixture for 4 h at70° C., it was cooled to room temperature and quenched with a slowaddition of MeOH (3 mL). The resulting reaction mixture was evaporatedto dryness, and purified by silica gel column chromatography (0-8% MeOHin CH₂Cl₂, v/v) to afford nucleoside 24a (6 mg, 72%) as a white solid.R_(f)=0.4 (10% MeOH in CH₂Cl₂, v/v). ESI-HRMS m/z 421.9883 ([M+H]⁺(C₁₂H₁₄N₅O₂ClI, calcd 421.9881). ¹H NMR (CDCl₃) δ 7.82 (s, 1H), 5.92 (s,2H), 5.60 (d, 1H, J=6.4 Hz), 4.78 (s, 1H), 4.68 (d, 1H, J=7.2 Hz),1.71-1.77 (m, 1H), 1.55 (s, 3H), 1.27 (s, 3H), 1.10-1.15 (m, 1H),0.98-1.02 (m, 1H).

(1′S,2′R,3′S,4′R,5′S)-4-(6-Amino-2-chloro-9H-purin-9-yl)-2,3-(O-isopropylidene)-1-[(di-tert-butylphosphate)dideuteromethyl]bicyclo[3.1.0]hexane(26a). Nucleoside 24a (6 mg, 17 μmol) was coevaporated with anhydroustoluene (3×10 mL), and dissolved in anhydrous THF (1 mL).Di-t-butyl-N,N′-diethylphosphoramidite (24 μL, 85 μmol) and tetrazole(12 mg, 169 μmol) were added. After stirring at rt for 4 h, the reactionmixture was cooled to −70° C. followed by the addition ofm-chloroperbenzoic acid (25 mg, 77%). The reaction mixture was warmed to0° C. and allowed to stir for 15 min, followed by the addition oftriethylamine (0.5 mL). The reaction mixture was evaporated to dryness,and the resulting crude residue was purified by silica gel columnchromatography (0-4% MeOH in CH₂Cl₂, v/v) to afford nucleoside 26a (7mg, 76%) as a white solid. R_(f)=0.3 (5% MeOH in CH₂Cl₂, v/v); ESI-HRMSm/z 546.2234 ([M+H]⁺ (C₂₃H₃₄D₂N₅O₆Cl, calcd 546.2217). ¹H NMR (MeOD-d₄)δ 8.15 (s, 1H), 5.34 (d, 1H, J=6.8 Hz), 4.98 (s, 1H), 4.77 (d, 1H, J=7.2Hz), 1.74-1.77 (m, 1H), 1.48-1.55 (m, 21H), 1.25 (s, 3H), 1.21-1.24 (m,1H), 1.09-1.13 (m, 1H).

(1′S,2′R,3′S,4′R,5′S)-4-(6-Amino-2-chloro-9H-purin-9-yl)-1-[phosphoryloxydideuteromethyl]-2,3-diol-bicyclo[3.1.0]hexane(6a). To a solution containing nucleoside 26a (7 mg, 12.8 μmol) in MeOHand H₂O (2 mL, 1:1, v/v) was added Dowex-50 resin (˜50 mg). The mixturewas stirred for 3 h at 70° C. and the resin removed by filtration. Thefiltrate was then treated with 1 M triethylammonium bicarbonate buffer(1 mL) and evaporated to dryness. The resulting mixture was purified bysemi preparative HPLC (retention time 16.5 min) to get 5′-monophosphate6a (1.62 mg, 32%) as white solid. ESI-HRMS m/z 392.0493 ([M−H]⁺(C₁₂H₁₂D₂N₅O₆ClP, calcd 392.0496). ¹H NMR (D₂O) δ 8.45 (s, 1H), 4.73 (s,1H), 3.97 (d, 1H, J=6.5 Hz), 1.72-1.77 (m, 1H), 1.39-1.43 (m, 1H),0.84-0.90 (m, 1H). ³¹P NMR (D₂O) δ 2.48.

Diethyl-(1′S,2′R,3′S,4′R,5′S)-4′-(6-chloro-2-iodo-purin-9-yl)-2′,3′-O-(isopropylidene)-bicyclo[3.1.0]hexanephosphonate (28a). Diisopropyl azodicarboxylate (86 μL, 0.44 mmol) wasadded at rt to a mixture of triphenylphosphine (115 mg, 0.44 mmol) and6-chloro-2-iodopurine (122 mg, 0.44 mmol) in anhydrous THF (4 mL). Afterstirring for 45 min, a solution of the compound 27a (70 mg, 0.22 mmol)in THF (4 mL) was added to the mixture. After stirring for 36 h, thereaction mixture was evaporated to dryness. The resulting residue waspurified by silica gel column chromatography (0-3% MeOH in CH₂Cl₂, v/v)to afford nucleoside 28a (89 mg, 70%) as a white solid. R_(f)=0.5 (5%MeOH in CH₂Cl₂, v/v); ESI-HRMS m/z 583.0374 [M+H]⁺, C₁₉H₂₅ClIN₄O₅P.H⁺:Calcd. 583.0373); ¹H NMR (CDCl₃) δ 8.64 (s, 1H), 5.39 (d, 1H, J=7.5 Hz),5.07 (s, 1H), 4.64 (d, 1H, J=7.5 Hz), 4.15-4.21 (m, 4H), 2.41 (t, 1H,J=16.0 Hz), 2.11-2.22 (m, 1H), 1.73-1.79 (m, 1H), 1.59-1.64 (m, 1H),1.54 (s, 3H), 1.35 (t, 3H, J=7.1 Hz), 1.28 (t, 3H, J=7.1 Hz), 1.25 (s,3H), 1.04-1.09 (m, 1H).

Diethyl-(1′S,2′R,3′S,4′R,5′S)-4′-(6-amino-2-iodopurin-9-yl)-2′,3′-O-(isopropylidene)-bicyclo[3.1.0]hexanephosphonate (29a). Nucleoside 28a (80 mg, 0.14 mmol) was treated with 2MNH₃ in i-PrOH (8 mL) and the mixture was heated to 70° C. and stirredfor 17 h. The reaction mixture was evaporated to dryness, and theresulting residue was purified by silica gel column chromatography (0-6%MeOH in CH₂Cl₂, v/v) to afford nucleoside 29a (48 mg, 64%) as a whitesolid. R_(f)=0.4 (7% MeOH in CH₂Cl₂, v/v); ESI-HRMS m/z 564.0873 [M+H]⁺,C₁₉H₂₇IN₅O₅P.H⁺: Calcd. 564.0856); ¹H NMR (CDCl₃) δ 8.15 (s, 1H), 5.74(s, 2H), 5.34 (d, 1H, J=6.1 Hz), 4.94 (s, 1H), 4.64 (d, 1H, J=6.1 Hz),4.12-4.19 (m, 4H), 2.19-2.42 (m, 2H), 1.69-1.74 (m, 1H), 1.55 (s, 3H),1.33 (t, 3H, J=7.1 Hz), 1.26 (t, 3H, J=7.1 Hz), 1.22 (s, 3H), 1.18-1.21(m, 1H), 1.03-1.09 (m, 1H).

(1′S,2′R,3′S,4′R,5′S)-4′-(6-Amino-2-iodoropurin-9-yl)-2′,3′-(dihydroxy)-1′-(phosphonomethylene)-bicyclo[3.1.0]hexane(7a). Nucleoside 29a (10 mg, 0.017 mmol) was coevaporated with anhydroustoluene (3×3 mL) and dissolved in anhydrous CH₂Cl₂ (2 mL). To thissolution was added iodotrimethylsilane (25 μl, 0.17 mmol). Afterstirring for 17 h, the reaction mixture was cooled to 0° C., followed bythe addition of ice-cold H₂O (20 mL) and CH₂Cl₂ (25 mL). The phases wereseparated and the aqueous phase washed with CH₂Cl₂ (2×35 mL) and diethylether (4×35 mL). The resulting aqueous phase was evaporated to drynessand purified by HPLC (retention time: 20 min) to afford 7a (4.1 mg, 49%)as a white solid. ESI-HRMS m/z 465.9777 [M−H]⁻, C₁₂H₁₄IN₅O₅P⁻: Calcd.465.9771); ¹H NMR (D₂O) δ8.19 (s, 1H), 4.71 (s, 1H), 4.58 (d, 1H, J=5.8Hz), 3.98 (d, 1H, J=5.8 Hz), 3.13 (q, 4H, J=7.2 Hz), 2.17 (t, 1H, 15.5Hz), 1.90 (t, 1H, 15.5 Hz), 1.68-1.74 (m, 1H), 1.35-1.42 (m, 1H), 1.21(t, 6H, J=7.2 Hz), 0.88-0.94 (m, 1H). ³¹P NMR (D₂O) δ 25.39. Purity >99%by HPLC (retention time: 5.9 min).

Diethyl-(1′S,2′R,3′S,4′R,5′S)-4′-(6-amino-2-iodopurin-9-yl)-2′,3′-(dihydroxy)-bicyclo[3.1.0]hexanephosphonate (16a). To a solution containing 29a (6 mg, 11.2 μmol) inMeOH:H₂O (2 mL, 1:1, v/v) was added Dowex-50 resin (˜50 Mg). The mixturewas stirred for 3 h at 70° C. and the resin removed by filtration.Filtrate was evaporated to dryness and resulting crude purified bysilica gel column chromatography (0-8% MeOH in CH₂Cl₂, v/v) to affordnucleoside 16a (4.5 mg, 49%) as a white solid. R_(f)=0.2 (5% MeOH inCH₂Cl₂, v/v); ESI-HRMS m/z 524.0562[M+H]⁺, C₁₆H₂₄IN₅O₅P.H⁺: Calcd.524.0560); ¹H NMR (MeOD-d₄) δ 8.25 (s, 1H), 4.77-4.81 (m, 2H), 4.10-4.19(m, 4H), 3.98 (d, 1H, J=6.8 Hz), 2.35-2.52 (m, 1H), 1.68-1.73 (m, 1H),1.49-1.52 (m, 1H), 1.29-1.35 (m, 6H), 0.85-0.91 (m, 1H).

Diethyl-(1′S,2′R,3′S,4′R,5′S)-4′-(6-amino-2-chloropurin-9-yl)-2′,3′-(hydroxy)-bicyclo[3.1.0]hexanephosphonate (9a). To a solution containing 30a (25 Mg, 0.053 mmol) inMeOH (3 mL) and water (3 mL) was added Dowex-50 resin (˜100 mg). Themixture was stirred for 3 h at 70° C. and the resin removed byfiltration. The filtrate was evaporated to dryness and resulting crudepurified by silica gel column chromatography (10% MeOH in EtOAc, v/v) toafford nucleoside 9a (8.3 mg, 40%) as a white solid. R_(f)=0.4 (10% MeOHin CH₂Cl₂, v/v); ESI-HRMS m/z 432.1197 [M+H]⁺, C₁₆H₂₃ClN₅O₅P.H⁺: Calcd.432.1204); ¹H NMR (CDCl₃) δ 8.31 (s, 1H), 4.73-4.79 (m, 2H), 4.06-4.18(m, 4H), 3.98 (d, J=6.6 Hz), 2.23-2.54 (m, 1H), 1.92-2.03 (m, 1H),1.72-1.78 (m, 1H), 1.51-1.57 (m, 1H), 1.28-1.35 (m, 6H), 0.80-0.89 (m,1H).

(1′S,2′R,3′S,4′R,5′S)-4′-(6-Amino-2-chloropurin-9-yl)-1′-[diisopropyl-(E)-ethenylphosphonate]-2,3-(dihydroxy)-bicyclo-[3.1.0]-hexane(11a). To a solution containing 38a (10 mg, 19.5 μmol) in MeOH:H₂O (4mL, 1:1, v/v) was added Dowex-50 resin (˜50 mg). The mixture was stirredfor 3 h at 70° C. and the resin removed by filtration. The filtrate wasevaporated to dryness and resulting crude purified by silica gel columnchromatography (0-10% MeOH in CH₂Cl₂, v/v) to afford nucleoside 11a (4.5mg, 49%) as a white solid. R_(f)=0.3 (10% MeOH in CH₂Cl₂, v/v); ESI-HRMSm/z 474.1679 [M+H]⁺, C₁₉H₃₀ClN₅O₅P.H⁺: Calcd. 474.1673); ¹H NMR(MeOD-d₄) δ 8.06 (s, 1H), 4.76 (d, 1H, J=6.9 Hz), 4.65-4.71 (m, 3H),4.07 (d, 1H, J=6.9 Hz), 3.67-3.72 (m, 1H), 3.55-3.59 (m, 1H), 2.10-2.16(m, 1H), 1.80-2.03 (m, 1H), 1.47-1.52 (m, 1H), 1.28-1.41 (m, 13H),0.66-0.72 (m, 1H).

Experimental Protocols for Biological Evaluation

CSQ mice and compound administration: Mice displaying the CSQ model ofsevere cardiomyopathy and heart failure were bred and maintainedaccording to a previously described method. The CSQ transgenic (TG) micewere originally provided by Dr. Larry Jones and developed hypertrophyfollowed by a lethal heart failure phenotype with death near the age of3 months.

Compound 3 and its analogues were dissolved in phosphate-bufferedsaline, pH=7.4 at 3.3 μM (200 μL total volume), filtered for sterilityfor in vivo administration at 6 μL per day for 28 days via amini-osmotic pump (Alzet) in the CSQ mice. Intact heart function in vivowas assessed by echocardiography following infusion of nucleotide- orvehicle

Mouse echocardiography: Transthoracic echocardiography was performedusing a linear 30-MHz transducer according to manufacturer'sinstructions (Vevo 660 High Resolution Imaging System from VisualSonics,Toronto, Canada) similar to previously described methods. Twodimensional-targeted M-mode echocardiographic measurements were carriedout at mid-papillary muscle level. Mice were anesthetized with 1%isoflurane using a vaporizer as previously described. Left ventricularend-diastolic (LVEDD) and end-systolic (LVESD) diameters, and FS(defined as LVEDD-LVESD/LVEDD) were measured. Parameters were measureddigitally on the M-mode tracings and were averaged from more than 3cardiac cycles.

Activation of human P2Y₁ receptors: Activity at the hP2Y₁ receptor wasquantified in 1321N1 human astrocytoma cells stably expressing thisreceptor, obtained from Prof T. K. Harden, University of North CarolinaSchool of Medicine, Chapel Hill, N.C. The procedure for measuringintracellular calcium using a FLIPR in response to nucleotidederivatives has been described. Cells were grown overnight in 100 ml ofmedium in 96-well flatbottom plates at 37° C. at 5% CO₂ or until theyreached ˜80% confluency. The calcium-4 assay kit (Molecular Devices,Sunnyvale, Calif.) was used as directed with no washing of cells. Cellswere loaded with 40 mL dye with probenecid in each well and incubatedfor 1 h at rt. The compound plate was prepared with dilutions of variouscompounds in Hank's Buffer at pH 7.2. Samples were run in duplicate witha FLIPR-Tetra (Molecular Devices) at rt. Cell fluorescence(excitation=485 nm; emission=525 nm) was monitored following exposure toa compound. Increases in intracellular calcium are reported as themaximum fluorescence value after exposure minus the basal fluorescencevalue before exposure.

Data analysis: Unless otherwise indicated, data were provided asmean±standard error of the mean. For analysis of multiple groups,one-way ANOVA and posttest comparison were used. Student's t-test forpaired or unpaired samples was used to evaluate the effects ofexperimental interventions; P<0.05 was taken as statisticallysignificant.

The terms “a” and “an” do not denote a limitation of quantity, butrather denote the presence of at least one of the referenced items. Theterm “or” means “and/or”. The terms “comprising”, “having”, “including”,and “containing” are to be construed as open-ended terms (i.e., meaning“including, but not limited to”). Recitation of ranges of values aremerely intended to serve as a shorthand method of referring individuallyto each separate value falling within the range, unless otherwiseindicated herein, and each separate value is incorporated into thespecification as if it were individually recited herein. The endpointsof all ranges are included within the range and independentlycombinable. All methods described herein can be performed in a suitableorder unless otherwise indicated herein or otherwise clearlycontradicted by context. The use of any and all examples, or exemplarylanguage (e.g., “such as”), is intended merely to better illustrate theinvention and does not pose a limitation on the scope of the inventionunless otherwise claimed. No language in the specification should beconstrued as indicating any non-claimed element as essential to thepractice of the invention as used herein.

Chemical compounds are described using standard nomenclature. Unlessdefined otherwise, all technical and scientific terms used herein havethe same meaning as is commonly understood by one of skill in the art towhich this invention belongs.

The Formulas include all subformulae thereof. For example Formulas I-VIinclude pharmaceutically acceptable salts, prodrugs and otherderivatives, hydrates, polymorphs, and thereof.

All forms (for example solvates, optical isomers, enantiomeric forms,polymorphs, free compound and salts) of an active agent may be employedeither alone or in combination.

In certain situations, the compounds of the Formulas may contain one ormore asymmetric elements such as stereogenic centers, including chiralcenters, stereogenic axes and the like, e.g. asymmetric carbon atoms, sothat the compounds can exist in different stereoisomeric forms. Thesecompounds can be, for example, racemates or optically active forms. Forcompounds with two or more asymmetric elements, these compounds canadditionally be mixtures of diastereomers. For compounds havingasymmetric centers, it should be understood that all of the opticalisomers and mixtures thereof are encompassed. In addition, compoundswith carbon-carbon double bonds may occur in Z- and E-forms, with allisomeric forms of the compounds being included in the present invention.Formulas I-VI include all chiral forms, stereoisomers, diastereomers,and enantiomers of compounds of Formulas I-VI.

The term “substituted”, unless otherwise indicated, means replacement ofone or more hydrogens with one or more substituents. Suitablesubstituents include, for example, hydroxyl, C₆-C₁₂ aryl, C₃-C₂₀cycloalkyl, C₁-C₂₀ alkyl, halogen, C₁-C₂₀ alkoxy, C₁-C₂₀ alkylthio,C₁-C₂₀ haloalkyl, C₆-C₁₂ haloaryl, pyridyl, cyano, thiocyanato, nitro,amino, C₁-C₁₂ alkylamino, C₁-C₁₂ aminoalkyl, acyl, sulfoxyl, sulfonyl,amido, or carbamoyl.

A dash (“-”) that is not between two letters or symbols is used toindicate a point of attachment for a substituent. For example,—(CH₂)C₃-C₇cycloalkyl is attached through carbon of the methylene (CH₂)group.

“Acyl” is an a group of the formula HC(O)—, alkyl-C(O)— orcycloalkyl-C(O)—, in which alkyl and cycloalkyl carry the definitionsset forth in this section. Acyl groups are covalently bound to theparent moiety via a single bond to the carbon of the acyl carbonyl.Non-limiting examples of suitable acyl groups include formyl, acetyl andpropanoyl.

“Alkyl” is a branched or straight chain saturated aliphatic hydrocarbongroup, having the specified number of carbon atoms, generally from 1 toabout 12 carbon atoms. The term C₁-C₄alkyl as used herein indicates analkyl group having from 1 to about 4 carbon atoms. Other embodimentsinclude alkyl groups having from 1 to 8 carbon atoms, 1 to 6 carbonatoms or from 1 to 2 carbon atoms, e.g., C₁-C₈ alkyl, C₁-C₆ alkyl, andC₁-C₂ alkyl.

“Alkenyl” is a straight or branched hydrocarbon chain comprising one ormore unsaturated carbon-carbon double bonds, which may occur in anystable point along the chain. Alkenyl groups described herein have theindicated number of carbon atoms. C₂-C₆ alkenyl indicates an alkenylgroup of from 2 to about 6 carbon atoms. When no number of carbon atomsis indicated, alkenyl groups described herein typically have from 2 toabout 12 carbon atoms, though lower alkenyl groups, having 8 or fewercarbon atoms, are preferred. Examples of alkenyl groups include ethenyl,propenyl, and butenyl groups.

“Alkynyl” is a straight or branched hydrocarbon chain comprising one ormore carbon-carbon triple bonds, which may occur in any stable pointalong the chain. Alkynyl groups described herein have the indicatednumber of carbon atoms. C₂-C₆ alkynyl indicates an alkynyl group of from2 to about 6 carbon atoms. When no number of carbon atoms is indicated,alkynyl groups described herein typically have from 2 to about 12carbons.

“Alkoxy” indicates an alkyl group as defined above with the indicatednumber of carbon atoms attached through an oxygen bridge (—O—). Examplesof alkoxy include, but are not limited to, methoxy, ethoxy, n-propoxy,i-propoxy, n-butoxy, 2-butoxy, t-butoxy, n-pentoxy, 2-pentoxy,3-pentoxy, isopentoxy, neopentoxy, n-hexoxy, 2-hexoxy, 3-hexoxy, and3-methylpentoxy. Alkoxy groups include, for example, methoxy groups.

“Alkylthio” indicates an alkyl group as defined above with the indicatednumber of carbon atoms attached through a sulfhydryl bridge (—SH—).Examples of alkylthio include, but are not limited to, methylthio,ethylthio, and isopropyl thio. Likewise “alkylsulfinyl” is an alkylgroup as defined above with the indicated number of carbon atomsattached through a sulfinyl bridge (—S(O)—) via a single covalent bondto the sulfur atom and “alkylsulfonyl” is a group attached through asulfonyl (—S(O)₂—) bridge.

“Aryl” indicates an aromatic group containing only carbon in thearomatic ring or rings. Such aromatic groups may be further substitutedwith carbon or non-carbon atoms or groups. Typical aryl groups contain 1or 2 separate, fused, or pendant rings and from 6 to about 12 ringatoms, without heteroatoms as ring members. Such substitution mayinclude fusion to a 5 to 7-membered saturated cyclic group thatoptionally contains 1 or 2 heteroatoms independently chosen from N, O,and S, to form, for example, a 3,4-methylenedioxy-phenyl group. Arylgroups include, for example, phenyl, naphthyl, including 1-naphthyl and2-naphthyl, and bi-phenyl.

In the term “(aryl)alkyl,” aryl and alkyl are as defined above, and thepoint of attachment to the parent moiety is on the alkyl group. Examplesof (aryl)alkyl groups include piperonyl and (phenyl)alkyl groups such asbenzyl, phenylethyl, and R-phenylisopropyl.

“Arylamino” is an aryl-NH— group. The arylamino group is covalentlybound to the parent moiety via a single bond from the nitrogen atom. Thenitrogen atom is optionally substituted. “Aryloxy” is an aryl-O— group.The aryloxy group is covalently bound to the parent moiety via a singlebond from the oxygen atom. “Arylsulfonyl” is an aryl-S(O₂)— group. Thebond to the parent moiety is through the sulfonyl.

“Cyano” is the radical —CN.

“Cycloalkyl” indicates saturated hydrocarbon ring groups, having thespecified number of carbon atoms, usually from 3 to about 8 ring carbonatoms, or from 3 to about 7 carbon atoms. Examples of cycloalkyl groupsinclude cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl as well asbridged or caged saturated ring groups such as norborane or adamantane.A bicyclic cycloalkyl” is a saturated bicyclic group having only carbonring atoms. Bicycloalkyl groups have 7 to 12 carbon ring atoms. Examplesof bicycloalkyl groups include s-endonorbornyl andcarbamethylcyclopentane.

“Cycloalkoxy” is a cycloalkyl-O—, wherein cycloalkyl is as definedabove. Cycloalkoxy groups include cyclopentyloxy.

“Halo” or “halogen” indicates fluoro, chloro, bromo, and iodo.

“Mono- and/or di-alkylamino” indicates secondary or tertiary alkyl aminogroups, wherein the alkyl groups are as defined above and have theindicated number of carbon atoms. The point of attachment of thealkylamino group is on the nitrogen. The alkyl groups are independentlychosen. Examples of mono- and di-alkylamino groups include ethylamino,dimethylamino, and methyl-propyl-amino.

“Mono- and/or dialkylaminoalkyl” groups are mono- and/or di-alkylaminogroups attached through an alkyl linker having the specified number ofcarbon atoms, for example a di-methylaminoethyl group. Tertiary aminosubstituents may by designated by nomenclature of the form N—R—N—R′,indicating that the groups R and R′ are both attached to a singlenitrogen atom

“Sulfonyl” is the bivalent radical —SO₂—.

“Thiol” is the radical —SH.

“Pharmaceutically acceptable salts” include derivatives of the disclosedcompounds in which the parent compound is modified by making inorganicand organic, non-toxic, acid or base addition salts thereof. The saltsof the present compounds can be synthesized from a parent compound thatcontains a basic or acidic moiety by conventional chemical methods.Generally, such salts can be prepared by reacting free acid forms ofthese compounds with a stoichiometric amount of the appropriate base(such as Na, Ca, Mg, or K hydroxide, carbonate, bicarbonate, or thelike), or by reacting free base forms of these compounds with astoichiometric amount of the appropriate acid. Such reactions aretypically carried out in water or in an organic solvent, or in a mixtureof the two. Generally, non-aqueous media like ether, ethyl acetate,ethanol, isopropanol, or acetonitrile are preferred, where practicable.Salts of the present compounds further include solvates of the compoundsand of the compound salts.

Examples of pharmaceutically acceptable salts include, but are notlimited to, mineral or organic acid salts of basic residues such asamines; alkali or organic salts of acidic residues such as carboxylicacids; and the like. The pharmaceutically acceptable salts include theconventional non-toxic salts and the quaternary ammonium salts of theparent compound formed, for example, from non-toxic inorganic or organicacids. For example, conventional non-toxic acid salts include thosederived from inorganic acids such as hydrochloric, hydrobromic,sulfuric, sulfamic, phosphoric, nitric and the like; and the saltsprepared from organic acids such as acetic, propionic, succinic,glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic,maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic,mesylic, esylic, besylic, sulfanilic, 2-acetoxybenzoic, fumaric,toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic,HOOC—(CH₂)_(n)—COOH where n is 0-4, and the like. Lists of additionalsuitable salts may be found, e.g., in Remington's PharmaceuticalSciences, 17th ed., Mack Publishing Company, Easton, Pa., p. 1418(1985). Other exemplary salts are amine salts of the phosphonic orphosphinic acid group including organic amine salts such astriethylamine salt, pyridine salt, picoline salt, ethanolamine salt,triethanolamine salt, dicyclohexylamine salt,N,N′-dibenzylethylenediamine salt, and the like; and amino acid saltssuch as arginate, asparginate, glutamate, and the like; and combinationscomprising one or more of the foregoing salts.

While the invention has been described with reference to a preferredembodiment, it will be understood by those skilled in the art thatvarious changes may be made and equivalents may be substituted forelements thereof without departing from the scope of the invention. Inaddition, many modifications may be made to adapt a particular situationor material to the teachings of the invention without departing fromessential scope thereof. Therefore, it is intended that the inventionnot be limited to the particular embodiment disclosed as the best modecontemplated for carrying out this invention, but that the inventionwill include all embodiments falling within the scope of the appendedclaims.

All cited patents, patent applications, and other references areincorporated herein by reference in their entirety.

ABBREVIATIONS

5′-AMP, adenosine 5′-monophosphate;

CSQ, calsequestrin;

DIBAL-H, diisobutylaluminium hydride;

DMEM, Dulbecco's modified Eagle medium;

FS, fractional shortening;

HEPES, N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid;

HPLC, high performance liquid chromatography;

HRMS, high resolution mass spectroscopy;

LV, left ventricular;

LVEDD, left ventricular end-diastolic diameter;

LVESD, left ventricular end-systolic diameter;

MRS2339,(1′S,2′R,3′S,4′R,5′S)-4-(6-amino-2-chloro-9H-purin-9-yl)-1-[phos-phoryloxymethyl]bicyclo[3.1.0]hexane-2,3-diol;

NS, normal saline;

PLC, phospholipase C;

SAR, structure activity relationship;

TBAF, tetrabutylammonium fluoride;

TBAP, tetrabutylammonium phosphate;

TBDPS-Cl, tert-butyl(chloro)diphenylsilane;

THF, tetrahydrofuran;

TEAA, triethylammonium acetate.

Schemes

Scheme 1: Reagents and Conditions: a) tert-Butylchlorodiphenylsilane,imidazole, DMAP, an. CH₂Cl₂, 93%; b) DIBAL-H, an. THF, 82%; c)Methanesulfonyl chloride, triethylamine, an. CH₂Cl₂, 96%; d) NaI, 65°C., an. 1,4-dioxane, 95%; e) Triethylphosphite, 110° C.; f) TBAF, THF,88%.

Scheme 2: Reagents and Conditions: a) Triphenylphosphine,2,6-dichloropurine, diisopropyl azodicarboxylate, an. THF, 75%; b) 2 MNH₃ in i-PrOH, 70° C., 80% for 9, 79% for 11; c) Iodotrimethylsilane,an. CH₂Cl₂, 23% for 4 and 27% for 5; d) Triphenylphosphine,6-chloropurine, diisopropyl azodicarboxylate, an. THF, 87%.

Scheme 3: Reagents and Conditions: a) Dess-Martin periodinane, an.CH₂Cl₂, 80%; b) Tetraisopropyl methylenediphosphonate, NaH, an. THF,83%; c) 2 M NH₃ in i-PrOH, 70° C., 80%; d) 10% Pd/C, H₂ (3 bar), MeOH:2Maq. NaOH (1:1, v/v), 79%; e) Iodotrimethylsilane, an. CH₂Cl₂, 47% for 10and 28% for 7; d) Triphenylphosphine, 6-chloropurine, diisopropylazodicarboxylate, an. THF, 87%.

Scheme 4: Reagents and Conditions: a) Dess-Martin periodinane, an.CH₂Cl₂, 72%; b) Tetraisopropyl methylenediphosphonate, NaH, an. THF,48%; c) TBAF, THF, 88%; d) Triphenylphosphine, 6-chloropurine,diisopropyl azodicarboxylate, an. THF, 85%; e) 2 M NH₃ in i-PrOH, 70°C., 85%; e) Iodotrimethylsilane, an. CH₂Cl₂, 78%.

Scheme 5: Reagents and Conditions: a) 10% Pd/C, H₂ (3 bar), MeOH, 72%;b) Triphenylphosphine, 2,6-dichloropurine, diisopropyl azodicarboxylate,an. THF, 40%; c) 2 M NH₃ in i-PrOH, 70° C., 71%; d) Iodotrimethylsilane,an. CH₂Cl₂, 45%.

Scheme 6: Reagents and Conditions: a) CBr₄, triphenylphosphine,triethylamine, 81%; b) Diethylmethylphosphite, 110° C., 95%; c) TBAF,THF, 91%, d) Triphenylphosphine, 2,6-dichloropurine, diisopropylazodicarboxylate, an. THF, 75%; e) 2 M NH₃ in i-PrOH, 70° C., 60%; f)Iodotrimethylsilane, an. CH₂Cl₂, 20% combined yield.

Scheme 7: A) Retrosynthetic analysis of 5′-phosphonate and 5′-methylphosphonates of (N)-methanocarba adenine or 2-Cl adenine derivatives. B)Retrosynthetic analysis of saturated and unsaturated long chain5′-phosphonates of (N)-methanocarba adenine or 2-Cl adenine derivatives.

Scheme 1a: Reagents and Conditions: a) 10% aqueous trifluoroacetic acid,60%; b) i) Thiophosphoryl chloride, 1,8-bis-(dimethylamino)naphthalene(proton sponge), pyridine, ii) Quenching the reaction withtetraethylammoniumbicorbonate (TEAB); c) i) Thiophosphoryl chloride,1,8-bis-(dimethylamino)naphthalene (proton sponge), pyridine, ii)Quenching the reaction with EtOH; d) Triphenylphosphine, I₂, imidazole,anhydrous THF, 74%, e) Dowex-50, MeOH:H₂O (1:1, v/v), 70° C.; f)Sodium-O,O-diethylthiophosphate, EtOH; THF; g) Trisodium thiophosphate,H₂O, 57%; h) Diethyl dithiophosphate potassium salt, DMF.

Scheme 2a: Reagents and Conditions: a) 2 M NH₃ in i-PrOH, 70° C., 68%;b) LiBD₄, anhydrous THF, 72%; c) i)Di-t-butylN,N′-diethylphosphoramidite, anhydrous THF, tetrazole; ii)m-chloroperbenzoic acid, 76%; d) Dowex-50 resin, MeOH:H₂O (1:1, v/v) 70°C.

Scheme 3a: Reagents and Conditions: a) Triphenylphosphine,6-chloro-2-iodopurine, diisopropyl azodicarboxylate, an. THF, 70%; b) 2M NH₃ in i-PrOH, 70° C., 64%; c) Iodotrimethylsilane, an. CH₂Cl₂, 49%for 7; d) Dowex-50, MeOH:H₂O (1:1, v/v), 70° C., 3 h, 49% for 16, e) i)Trimethylsilylacetylene, Pd(Ph₃)₄, CuI, TEA, anhydrous DMF ii) TBAF,anhydrous THF.

Scheme 4a: Reagents and Conditions: a) Dess-Martin periodinane, an.CH₂Cl₂; b) Tetraethyl methylenediphosphonate, NaH, an. THF; c) 2 M NH₃in i-PrOH, 70° C.; d) O-nitrobenzenesulfonylhydrazide, Et₃N, CH₂Cl₂; e)Dowex-50, MeOH:H₂O (1:1, v/v), 70° C.

TABLE 1 Phosphonate analogues: structure and effects on in vivo heartfunction as determined by echocardiography-derived FS in CSQ heartfailure mice. FS in % in CSQ No Structure Mice^(a) n =  3

15.47 ± 1.15 10  4

20.25 ± 1.19  8  5

16.23 ± 0.93 13  7

12.12 ± 1.2 11  8

13.88 ± 2.12  8  9

19.26 ± 1.23 16 10

11.15 ± 1.44 12 11

 15.0 ± 1.2 10 12

ND ^(a)at 3.3 μM. The vehicle control mice displayed a % FS of 13.78 ±1.19% (n = 16). ND- not determined.

TABLE 2 Novel phosphate and phosphonate analogues: structure and effectson in vivo heart function as determined by echocardiography-derived FSin CSQ heart failure mice. FS in % in CSQ No Structure Mice^(a) n =Charged nucleotide  4a

14.33 ± 3.77 4  5a

 6a

 7a

10.33 ± 1.77 5 Masked nucleotides  8a

 9a

 8.58 ± 2.09 4 10a

11a

12.94 ± 0.98 6 12a

13a

14a

15a

16a

ND, No effect ex- pected 17a

^(a)at 10 μM. The vehicle control mice displayed a % FS of 7.13 ± 1.49%(n = 4). The CSQ mice used here were phenotypically more severe than themice used for data in Table 1.² ND- not determined.

1. A phosphonate or phosphinate N-methanocarba derivative of AMPcomprising,

wherein Q¹ is O or S; R¹ is hydrogen, optionally substituted alkyl,optionally substituted cycloalkyl, halogen, or N(R⁶)₂, wherein each R⁶is independently hydrogen, optionally substituted alkyl, or optionallysubstituted cycloalkyl; R² is hydrogen, optionally substituted alkyl,optionally substituted cycloalkyl, optionally substituted alkynyl,N(R⁶)₂, or halogen; R³ is hydrogen, optionally substituted alkyl,N(R⁶)₂, or halogen; R⁴ is hydroxyl, optionally substituted alkyl,optionally substituted alkoxy, optionally substituted aryl, optionallysubstituted —Oaryl, or N(R⁶)₂; R⁵ is hydroxyl, optionally substitutedalkyl, optionally substituted alkoxy, optionally substituted aryl, oroptionally substituted —Oaryl; or alternatively, R⁴ and R⁵ form a 5- or6-membered cyclic structure with the phosphorus atom where the cyclicstructure contains at least two oxygen atoms and at least 2 or 3 carbonatoms, wherein the carbon atoms are optionally substituted with alkyl oraryl where the chain is attached; and Y is a linking group linked to thephosphorus atom by a carbon atom; or

wherein X is O or S; n is 1, 2, or 3; and R⁷ is optionally substitutedalkyl or optionally substituted aryl; or

wherein Z is a bond or —O—C(═O)— where the carbonyl carbon is bonded tothe oxygen of the bicycle group and the oxygen is bonded to thephosphorus atom; or

wherein R⁸ is hydrogen or optionally substituted alkyl; and R⁸ isoptionally substituted alkyl, optionally substituted alkoxy, oroptionally substituted aryl; or

wherein G is O or S—S; and R¹⁰ is hydrogen, hydroxyl, optionallysubstituted alkyl, optionally substituted alkoxy, or optionallysubstituted aryl; or

wherein R¹¹ is hydrogen, optionally substituted alkyl, or optionallysubstituted aryl; or

wherein Q¹ is O or S; Q² is O or S; R¹ is hydrogen, optionallysubstituted alkyl, optionally substituted cycloalkyl, halogen, orN(R⁶)₂, wherein each R⁶ is independently hydrogen, optionallysubstituted alkyl, or optionally substituted cycloalkyl; R² is hydrogen,optionally substituted alkyl, optionally substituted cycloalkyl,optionally substituted alkynyl; N(R⁶)₂, or halogen; R³ is hydrogen,optionally substituted alkyl, N(R⁶)₂, or halogen; R⁴ is hydroxyl,optionally substituted alkyl, optionally substituted alkoxy, optionallysubstituted aryl, optionally substituted —Oaryl, or N(R⁶)₂; R⁵ ishydroxyl, optionally substituted alkyl, optionally substituted alkoxy,optionally substituted aryl, or optionally substituted —Oaryl; oralternatively, R⁴ and R⁵ form a 5- or 6-membered cyclic structure withthe phosphorus atom where the cyclic structure contains at least twooxygen atoms and at least 2 or 3 carbon atoms, wherein the carbon atomsare optionally substituted with alkyl or aryl where the chain isattached; and Y¹ is a linking group, with the proviso that when Q¹ andQ² are both O, and Formula (VII) is not enriched with deuterium, then R⁴and R⁵ are not both hydroxyl, a deuterium enriched isomer thereof, or apharmaceutically acceptable salt thereof.
 2. The phosphonate orphosphinate N-methanocarba derivative of AMP of claim 1, comprisingFormula (I) or (VII).
 3. The phosphonate or phosphinate N-methanocarbaderivative of AMP of claim 1, Formula (I) wherein Q¹ is O; R¹ is N(R⁶)₂wherein each R⁶ is hydrogen; R² is halogen; R³ is hydrogen; R⁴ ishydroxyl or optionally substituted alkoxy; R⁵ is hydroxyl or optionallysubstituted alkoxy; and Y is a linking group linked to the phosphorusatom by a carbon atom.
 4. The phosphonate or phosphinate N-methanocarbaderivative of AMP of claim 1, Formula (I) wherein Q¹ is O; R¹ is N(R⁶)₂wherein each R⁶ is hydrogen; R² is halogen; R³ is hydrogen; R⁴ ishydroxyl or optionally substituted alkoxy; R⁵ is hydroxyl or optionallysubstituted alkoxy; and Y is a C₁-C₆ alkylene.
 5. The phosphonate orphosphinate N-methanocarba derivative of AMP of claim 1, Formula (VII)wherein Q¹ is O; Q² is S; R¹ is N(R⁶)₂ wherein each R⁶ is hydrogen; R²is halogen; R³ is hydrogen; R⁴ is hydroxyl or optionally substitutedalkoxy; R⁵ is hydroxyl or optionally substituted alkoxy; and Y¹ is aC₁-C₆ alkylene.
 6. The phosphonate or phosphinate N-methanocarbaderivative of AMP of claim 1, Formula (VII) wherein Q¹ is S; Q² is O; R¹is N(R⁶)₂ wherein each R⁶ is hydrogen; R² is halogen; R³ is hydrogen; R⁴is hydroxyl or optionally substituted alkoxy; R⁵ is hydroxyl oroptionally substituted alkoxy; and Y¹ is a C₁-C₆ alkylene.
 7. Thephosphonate or phosphinate N-methanocarba derivative of AMP of claim 1,comprising


8. The phosphonate or phosphinate N-methanocarba derivative of AMP ofclaim 1, comprising


9. A pharmaceutical composition comprising the phosphonate orphosphinate N-methanocarba derivative of AMP of claim 1 and apharmaceutically acceptable excipient.
 10. A method of treating amammalian subject in need of treatment for a cardiac or vascular diseaseor condition responsive to activation of the cardiac and/or vascular P2Xreceptor, comprising administering an effective amount of a phosphonateor phosphinate N-methanocarba derivative of AMP for the treatment forthe cardiac or vascular disease or condition responsive to activation ofthe cardiac and/or vascular P2X receptor.
 11. The method of claim 10,wherein the phosphonate or phosphinate N-methanocarba derivative of AMPis an ester prodrug analog.
 12. The method of claim 10, wherein thephosphonate or phosphinate N-methanocarba derivative of AMP is inaccordance with claim
 1. 13. A method of improving cardiac contractileperformance or cardiac function in a mammal in need thereof, comprisingadministering an effective amount of phosphonate or phosphinateN-methanocarba derivative of AMP for the treatment for the improvementof cardiac contractile performance or cardiac function.
 14. The methodof claim 13, wherein the phosphonate or phosphinate N-methanocarbaderivative of AMP is an ester prodrug analog.
 15. The method of claim13, wherein the phosphonate or phosphinate N-methanocarba derivative ofAMP is in accordance with claim
 1. 16. A method of treating a mammaliansubject in need of treatment for a cardiac hypertrophy, systolic heartfailure, diastolic heart failure, ischemic cardiomyopathy, non-ischemiccardiomyopathy, or adverse cardiac remodeling following injury to theheart as a result of ischemia/reperfusion or non-ischemic causescomprising administering an effective amount of phosphonate orphosphinate N-methanocarba derivative of AMP.
 17. The method of claim16, wherein the phosphonate or phosphinate N-methanocarba derivative ofAMP is an ester prodrug analog.
 18. The method of claim 16, wherein thephosphonate or phosphinate N-methanocarba derivative of AMP is inaccordance with claim 1.